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The recent development of whole slide imaging (wsi) is rapidly making many advances in pathology and enabling the creation of novel tools and applications for the pathology community, including virtual microscopy, and computer - aided quantification and diagnosis . In contrast to conventional light microscopy that allows only a view of a fraction of a specimen at a time, wsi offers a digital replica of an entire histopathology slide . The data can be processed with a pattern recognition algorithm to identify features of interest . This technique, called whole slide pattern recognition, can provide more information than traditional pathology slide analysis, minimizes tedious visual inspection by trained pathologists and has the potential to improve clinical diagnosis . First of all, a typical wsi dataset can contain a number of pixels at tera - scale (10) and previous computational methods used to identify targets on traditional histopathology images, including texture - analysis, nuclei pattern classification, and edge detection, are impractical for wsi due to computational time requirements . Parallel processing strategies have been developed to handle wsi; however, boundary artifacts can jeopardize the integration of the analysis results and introduce a substantial amount of re - processing time . In addition, the abundant information provided by the whole slide analysis also requires a method to provide a quantitative assessment and to present meaningful results in a manner that is easy to interpret . In this paper, we report a wsi analysis method that conducts automatic stain recognition to estimate the density distribution of the recognized stains across an entire pathology slide . The whole slide images were tessellated into smaller image blocks with overlapping regions at the image boundary to enable parallel processing . Pixel - based stain recognition with morphological smoothing was applied to identify stains in the tessellated images . The density distribution of the identified stains was calculated by the kernel density estimator, yielding a distribution image of a feature or a particular cell type labeled by the stains . To validate our method, we applied it to specimen slides from an ongoing study using a rat cardiac allograft rejection model and also another study using a rat heart ischemia / reperfusion injury model . In both studies, the macrophage infiltration was revealed by immunohistochemistry (ihc) with anti - rat ed1 antibody . The accuracy of the stain recognition algorithm was first validated by comparing its results with manual counting without involving the wsi technique . The pathology images were obtained with a standard light microscope and the results of our stain recognition algorithm were compared with those obtained from manual counting . Then, our method was further validated / correlated with the results obtained from in - vivo cellular magnetic resonance imaging (mri), an imaging technique that can detect macrophage infiltration when labeled with ultrasmall superparamagnetic iron - oxide (uspio) or micron - sized iron - oxide (mpio) particles . Our method was also validated / correlated with the results obtained from ex - vivo cellular mri, which were conducted using magnetic resonance microscopy (mrm) to achieve a much higher resolution for our final examination . To demonstrate the potential utilities of our method, we trained the algorithm to recognize different targets under hematoxylin and eosin (h and e) stain and to present their distribution across the tissue sections . We further calculated the diameter of the recognized targets and estimated the spatial distribution of cell nuclear diameter, which may have potential application in characterizing tissue in histopathology slides . The rat cardiac allograft rejection model, as described in a study, was established using dark agouti and brown norway rats (harlan, indianapolis, in) as the donor - recipient transplantation pairs . The superior vena cava of the graft heart was anastomosed to the recipient inferior vena cava (ivc) and the aorta of the graft heart was anastomosed to the recipient abdominal aorta, with the recipient proximal ivc partially obstructed to increase the pre - load . The graft, with intact pulmonary circulation, received proper pressure and volume loading without detectable atrophy over time . On day 7 after the transplantation, the graft hearts were scanned using in - vivo cellular mri and then harvested for pathology inspections . A suture was tied around the left anterior descending artery for 45 min and then was cut to allow reperfusion . After 2 days, the heart was harvested and scanned by ex - vivo mrm . Cellular mri experiments were conducted by using dextran - coated uspio particles or polystyrene - coated mpio particles (bangs laboratories, fishers, in) as an mri contrast agent to image macrophage distribution . The iron - oxide particles are taken up by macrophages in circulation and create signal - voids in t2 * -weighted images to reveal the macrophage infiltration in - vivo, offering a unique way to correlated the ihc stain distribution obtained from our method . In our study, the iron - oxide particles were given to the animals before surgery . The rats, undergoing heterotopic heart transplantation, received uspio particles and were scanned by in - vivo cellular mri . The in - vivo mri scan was conducted on a bruker 7-t scanner (bruker, billerica ma). The rats were intubated and ventilated during the mri scan and electrocardiogram (ecg) leads were placed on the abdomen using a gating and monitoring system (sa instruments, stony brook, ny). T2 * -weighted magnetic resonance (mr) images, an imaging modality known to be sensitive to the signal void artifact created by iron - oxide particles, were acquired using a flash sequence with ecg and respiratory gating . Tr = the respiration cycle (~1 s), te = 5 ms, field of view (fov) = 4 cm, slice thickness = 1.5 mm, in - plane resolution = 156 m . The ex - vivo mri scans were conducted using a bruker 11.7-tesla scanner (bruker, billerica ma). T2 * -weighted mr images were also acquired using a flash sequence with te = 5.8 ms, fov = 1.2 cm, and isotropic resolution = 23 m . The hearts were fixed in 4% paraformaldehyde for 1 - 2 weeks and embedded in paraffin . #dc2002), which streamed slides in preheated high ph target retrieval solution (dako #s3308, carpinteria, ca, usa) for 15 min . This was followed by cooling of the slides at room temperature for 20 min and rinsing the slides in distilled water before commencing the staining steps . To stain the slides, the tissue sections were incubated with 10% horse serum for 10 min at room temperature to block non - specific binding . The slides were then incubated with 1:200 anti - rat ed1 monoclonal purified immunoglobulin g (abd serotec, oxford, uk) for 2 h. after pbs wash, the slides were incubated with biotinylated secondary antibodies (lsab kit, dako, carpinteria, ca, usa) for 30 min and labeled with the streptavidin - horseradish peroxidase (dako, carpinteria, ca, usa) for 30 min . Then the slides were stained with aminoethyl carbazole chromogen (skytek laboratories, inc ., a rat spleen tissue section was used as a positive control section, whereas a normal rat cardiac tissue was used as a negative control section . The stained slides were scanned on a whole slide scanner (nanozoomer 2.0-ht, hamamatsu, japan) to acquire wsi at 20 magnification . The resulting pathology images were then processed by our whole slide pattern recognition method, as detailed in the following section . The whole slide image was tessellated to enable the whole slide pattern recognition, as shown in figure 1 . The tessellation lines divide the whole slide image into several n - by - n sized blocks and the choice of n depends on the application scenario and the applied recognition algorithm . A higher value offers a larger field of view, but it may exceed the maximum allowable image size for the algorithm . After tessellation, each block was padded with an additional margin, m - pixel wide, to the neighboring block . The padding setting was devised for accurate accounting of stains near the block boundary, since a strict boundary could bisect a stain, incorrectly assigning it to both neighboring blocks . The choice of m considers the span of individual stain and requires prior information about their morphology . An m - pixel - wide margin allows stains with a maximum span of m to be fully recognized within the tessellated image . In our study, we used n = 1000 and the value of m was set to 100 . The tessellation scheme for the whole slide pattern recognition . The whole slide image is tessellated into n - by - n sized blocks to enable parallel recognition . Each block is patched with additional m - width margin to consider stains bisected by the tessellation boundary the center of each recognized stain can be used to determine whether it should be counted or ignored to avoid double counting of stains, as illustrated by the example scenario in figure 2 . One should note that the tessellation borders are present at all sides of the tessellated image and figure 2 shows only the border between two adjacent images blocks to simplify the illustration . As shown in figure 2a, our stain recognition algorithm is applied to the image block to the left and the recognized stains with center points inside the tessellation line (annotated by yellow) are counted, whereas those outside the line (annotated by red) are ignored . Similarly, in figure 2b, our stain recognition algorithm is applied to the image block to the right . The recognized stains with center points inside the tessellation line (annotated by yellow) are counted, whereas the rest are ignored (annotated by red). (a) the stain recognition is applied to the image block to the left to recognize the stains . The center locations of the stains (marked by dot points) are used to determine whether a recognized stain should be counted (annotated by yellow) or ignored (annotated by red). (b) the stain recognition is applied to the image block to the right, showing how double counting is avoided . The counted stains (annotated by yellow) have center points located inside the tessellation line the flow chart of our stain recognition algorithm is shown in figure 3 . The method is composed of three modules, including a classification module that performs pixel - wise color classification, a morphology smoothing module that eliminates the fragments considered to be false positives and an analysis module that identifies each isolated stain region . It is noteworthy that a variety of stain recognition algorithms can be applied to this problem, and in this study, we have used the simplest pixel - wise color recognition approach for its low computation time and stable performance . The red - green - blue color channels were used as the classification features and the output (classification result) was either 0 (background) or 1 (foreground). The stain classification was conducted using nearest neighbor method, a popular method in data mining . Nearest neighbor method required a training dataset of background and foreground stain image . In this study, we manually cropped the background and foreground stain from the wsi data as the training dataset . Alternatively, one may also get training data from the positive and negative controls . The nearest neighbor method classified each image pixel based on its color difference with the pixels in the training images . If the minimum difference was from the foreground stain, then the pixel was classified as foreground; otherwise, it was classified as background . This classification was applied to each pixel in the input image [figure 3b] to obtain the binary recognition output [figure 3c]. This output image might have small fragments due to error classification or the partial slice coverage of the stained cells . To eliminate fragments in the binary images and to facilitate analysis, morphological opening and closing were applied 3 times using a 3-by-3 square structuring element to smooth the contours of the recognized stains, as shown in figure 3d . Finally, the connected component analysis identified each isolated stain region in the image and the center location of each stain was calculated for further density estimation . The pixel - wise classification is trained by a foreground stain image and also a background stain image (a). Each pixel of the input image (b) is then classified using nearest neighbor classification to obtain a binary image (c). The small fragments in the binary image are then eliminated by morphological closing and opening, resulting in a smoother output (d). This image is then analyzed by connected component analysis to obtain the center locations of the recognized stains since the locations of the recognized stains were discrete points scattered across the wsi, a regression analysis method was used to calculate their density distribution . This was carried out by the kernel density estimator as shown in figure 4, where figure 4a shows an exemplary result of the discrete stain locations and figure 4b shows their density distribution estimated by the kernel density estimator . We had a total of k stains obtained from our whole slide pattern recognition and each of them was located at coordinate xi . The kernel density estimator estimated the density distribution function by using the following formulae: the exemplary analysis result of the kernel density estimator . (b) their density distribution can be estimated by the kernel density estimator where h is the kernel bandwidth and is the kernel function . The scalar feature (e.g. Diameter or area) of the k recognized stains can also be estimated using kernel regression . Where yi is a scalar feature of the i stain . Equation 2 can be used to estimate the spatial distribution of the feature across the entire pathology slide . The bandwidth of the kernel determines the variability of the density estimation . A higher value of the bandwidth generates a smoothing effect, leading to reduced variability . On the contrary, a lower value creates a sharper estimation, but may cause over - fitting and larger variability . In this study, we used a data - driven method to automatically determine the bandwidth . We used equation 1 to estimate the ed1 stain distribution images and equation 2 to estimate the spatial distribution of the stain feature . The source codes of our whole slide recognition program (ws recognizer) are publicly available at http://ws-recognizer.labsolver.org . The validation was conducted using a total of 4 rats from the acute cardiac allograft rejection study in our laboratory . For each rat, one tissue section was obtained from the mid portion of the excised heart . For each slide, 16 pathology images were acquired on a transmitted light microscope (olympus proves ax 70) at 40 objective lens . The field of view covered different areas in the slide to acquire images with different macrophage density . In each pathology image, manual counting and automatic stain recognition were conducted separately to avoid a spurious correlation between the manual counting and the automated recognition . Manual counting was conducted without knowing the stain recognition results and the training data for automatic recognition were selected without knowledge of the manual - counting results . The final comparison was conducted by performing regression analysis to calculate the correlation coefficient . To validate our method on wsi data, we compared the ed1 stain distribution image with both in - vivo and ex - vivo cellular mr images . To facilitate a visual comparison, the orientations of the mr images were manually rotated to match those of the pathology images . The hypointensity patches shown in the t2 * -weighted images were due to the accumulation of the iron - oxide labeled macrophages, a feature that allowed us to examine whether our ed1 stain distribution image was in good agreement with the distribution shown on cellular mri . The rat cardiac allograft rejection model, as described in a study, was established using dark agouti and brown norway rats (harlan, indianapolis, in) as the donor - recipient transplantation pairs . The superior vena cava of the graft heart was anastomosed to the recipient inferior vena cava (ivc) and the aorta of the graft heart was anastomosed to the recipient abdominal aorta, with the recipient proximal ivc partially obstructed to increase the pre - load . The graft, with intact pulmonary circulation, received proper pressure and volume loading without detectable atrophy over time . On day 7 after the transplantation, the graft hearts were scanned using in - vivo cellular mri and then harvested for pathology inspections . A suture was tied around the left anterior descending artery for 45 min and then was cut to allow reperfusion . After 2 days, the heart was harvested and scanned by ex - vivo mrm . Cellular mri experiments were conducted by using dextran - coated uspio particles or polystyrene - coated mpio particles (bangs laboratories, fishers, in) as an mri contrast agent to image macrophage distribution . The iron - oxide particles are taken up by macrophages in circulation and create signal - voids in t2 * -weighted images to reveal the macrophage infiltration in - vivo, offering a unique way to correlated the ihc stain distribution obtained from our method . In our study, the iron - oxide particles were given to the animals before surgery . The rats, undergoing heterotopic heart transplantation, received uspio particles and were scanned by in - vivo cellular mri . The in - vivo mri scan was conducted on a bruker 7-t scanner (bruker, billerica ma). The rats were intubated and ventilated during the mri scan and electrocardiogram (ecg) leads were placed on the abdomen using a gating and monitoring system (sa instruments, stony brook, ny). T2 * -weighted magnetic resonance (mr) images, an imaging modality known to be sensitive to the signal void artifact created by iron - oxide particles, were acquired using a flash sequence with ecg and respiratory gating . Tr = the respiration cycle (~1 s), te = 5 ms, field of view (fov) = 4 cm, slice thickness = 1.5 mm, in - plane resolution = 156 m . The ex - vivo mri scans were conducted using a bruker 11.7-tesla scanner (bruker, billerica ma). T2 * -weighted mr images were also acquired using a flash sequence with te = 5.8 ms, fov = 1.2 cm, and isotropic resolution = 23 m . The hearts were fixed in 4% paraformaldehyde for 1 - 2 weeks and embedded in paraffin . #dc2002), which streamed slides in preheated high ph target retrieval solution (dako #s3308, carpinteria, ca, usa) for 15 min . This was followed by cooling of the slides at room temperature for 20 min and rinsing the slides in distilled water before commencing the staining steps . To stain the slides, the tissue sections were incubated with 10% horse serum for 10 min at room temperature to block non - specific binding . The slides were then incubated with 1:200 anti - rat ed1 monoclonal purified immunoglobulin g (abd serotec, oxford, uk) for 2 h. after pbs wash, the slides were incubated with biotinylated secondary antibodies (lsab kit, dako, carpinteria, ca, usa) for 30 min and labeled with the streptavidin - horseradish peroxidase (dako, carpinteria, ca, usa) for 30 min . Then the slides were stained with aminoethyl carbazole chromogen (skytek laboratories, inc ., a rat spleen tissue section was used as a positive control section, whereas a normal rat cardiac tissue was used as a negative control section . The stained slides were scanned on a whole slide scanner (nanozoomer 2.0-ht, hamamatsu, japan) to acquire wsi at 20 magnification . The resulting pathology images were then processed by our whole slide pattern recognition method, as detailed in the following section . The whole slide image was tessellated to enable the whole slide pattern recognition, as shown in figure 1 . The tessellation lines divide the whole slide image into several n - by - n sized blocks and the choice of n depends on the application scenario and the applied recognition algorithm . A higher value offers a larger field of view, but it may exceed the maximum allowable image size for the algorithm . After tessellation, each block was padded with an additional margin, m - pixel wide, to the neighboring block . The padding setting was devised for accurate accounting of stains near the block boundary, since a strict boundary could bisect a stain, incorrectly assigning it to both neighboring blocks . The choice of m considers the span of individual stain and requires prior information about their morphology . An m - pixel - wide margin allows stains with a maximum span of m to be fully recognized within the tessellated image . In our study, we used n = 1000 and the value of m was set to 100 . The tessellation scheme for the whole slide pattern recognition . The whole slide image is tessellated into n - by - n sized blocks to enable parallel recognition . Each block is patched with additional m - width margin to consider stains bisected by the tessellation boundary the center of each recognized stain can be used to determine whether it should be counted or ignored to avoid double counting of stains, as illustrated by the example scenario in figure 2 . One should note that the tessellation borders are present at all sides of the tessellated image and figure 2 shows only the border between two adjacent images blocks to simplify the illustration . As shown in figure 2a, our stain recognition algorithm is applied to the image block to the left and the recognized stains with center points inside the tessellation line (annotated by yellow) are counted, whereas those outside the line (annotated by red) are ignored . Similarly, in figure 2b, our stain recognition algorithm is applied to the image block to the right . The recognized stains with center points inside the tessellation line (annotated by yellow) are counted, whereas the rest are ignored (annotated by red). (a) the stain recognition is applied to the image block to the left to recognize the stains . The center locations of the stains (marked by dot points) are used to determine whether a recognized stain should be counted (annotated by yellow) or ignored (annotated by red). (b) the stain recognition is applied to the image block to the right, showing how double counting is avoided . The counted stains (annotated by yellow) have center points located inside the tessellation line the flow chart of our stain recognition algorithm is shown in figure 3 . The method is composed of three modules, including a classification module that performs pixel - wise color classification, a morphology smoothing module that eliminates the fragments considered to be false positives and an analysis module that identifies each isolated stain region . It is noteworthy that a variety of stain recognition algorithms can be applied to this problem, and in this study, we have used the simplest pixel - wise color recognition approach for its low computation time and stable performance . The red - green - blue color channels were used as the classification features and the output (classification result) was either 0 (background) or 1 (foreground). The stain classification was conducted using nearest neighbor method, a popular method in data mining . Nearest neighbor method required a training dataset of background and foreground stain image . In this study, we manually cropped the background and foreground stain from the wsi data as the training dataset . The nearest neighbor method classified each image pixel based on its color difference with the pixels in the training images . If the minimum difference was from the foreground stain, then the pixel was classified as foreground; otherwise, it was classified as background . This classification was applied to each pixel in the input image [figure 3b] to obtain the binary recognition output [figure 3c]. This output image might have small fragments due to error classification or the partial slice coverage of the stained cells . To eliminate fragments in the binary images and to facilitate analysis, morphological opening and closing were applied 3 times using a 3-by-3 square structuring element to smooth the contours of the recognized stains, as shown in figure 3d . Finally, the connected component analysis identified each isolated stain region in the image and the center location of each stain was calculated for further density estimation . The pixel - wise classification is trained by a foreground stain image and also a background stain image (a). Each pixel of the input image (b) is then classified using nearest neighbor classification to obtain a binary image (c). The small fragments in the binary image are then eliminated by morphological closing and opening, resulting in a smoother output (d). This image is then analyzed by connected component analysis to obtain the center locations of the recognized stains since the locations of the recognized stains were discrete points scattered across the wsi, a regression analysis method was used to calculate their density distribution . This was carried out by the kernel density estimator as shown in figure 4, where figure 4a shows an exemplary result of the discrete stain locations and figure 4b shows their density distribution estimated by the kernel density estimator . We had a total of k stains obtained from our whole slide pattern recognition and each of them was located at coordinate xi . The kernel density estimator estimated the density distribution function by using the following formulae: the exemplary analysis result of the kernel density estimator . (b) their density distribution can be estimated by the kernel density estimator where h is the kernel bandwidth and is the kernel function . The scalar feature (e.g. Diameter or area) of the k recognized stains can also be estimated using kernel regression . Where yi is a scalar feature of the i stain . Equation 2 can be used to estimate the spatial distribution of the feature across the entire pathology slide . A higher value of the bandwidth generates a smoothing effect, leading to reduced variability . On the contrary, a lower value creates a sharper estimation, but may cause over - fitting and larger variability . In this study, we used a data - driven method to automatically determine the bandwidth . We used equation 1 to estimate the ed1 stain distribution images and equation 2 to estimate the spatial distribution of the stain feature . The source codes of our whole slide recognition program (ws recognizer) are publicly available at http://ws-recognizer.labsolver.org . The validation was conducted using a total of 4 rats from the acute cardiac allograft rejection study in our laboratory . For each rat, one tissue section was obtained from the mid portion of the excised heart . For each slide, 16 pathology images were acquired on a transmitted light microscope (olympus proves ax 70) at 40 objective lens . The field of view covered different areas in the slide to acquire images with different macrophage density . In each pathology image, manual counting and automatic stain recognition were conducted separately to avoid a spurious correlation between the manual counting and the automated recognition . Manual counting was conducted without knowing the stain recognition results and the training data for automatic recognition were selected without knowledge of the manual - counting results . To validate our method on wsi data, we compared the ed1 stain distribution image with both in - vivo and ex - vivo cellular mr images . To facilitate a visual comparison, the orientations of the mr images were manually rotated to match those of the pathology images . The hypointensity patches shown in the t2 * -weighted images were due to the accumulation of the iron - oxide labeled macrophages, a feature that allowed us to examine whether our ed1 stain distribution image was in good agreement with the distribution shown on cellular mri . The sample result of the stain recognition algorithm is shown in figure 5a, where the recognized stains are annotated by the yellow contour . The training images are presented in figure 5b and c, which are the background and foreground ihc stain images, respectively . The image shows the infiltration of ed1 macrophages due to acute cardiac allograft transplant rejection and the brownish ihc stains were identified by our recognition method . The total numbers of macrophages were counted by summing up the number of annotated regions in the image and the center point of each annotated region can be calculated to facilitate further stain density estimation . The result of our stain recognition and the training images used in the stain classification . (a) the stain recognition is applied to a pathology image of myocardium under acute cardiac allograft rejection, where macrophage accumulation revealed by immunohistochemistry (ihc) stains can be recognized by our algorithm . The stain recognition algorithm is trained by using one background image (b) and one foreground ihc stain image (c) and the recognized stains are annotated by yellow to illustrate the performance of our algorithm the result of the manual validation is shown in figure 6, where the numbers of ihc stains counted by our automated stain recognition algorithm were correlated with manual counting . The analysis shows an r = 0.8029 and a correlation coefficient of 0.8961 between the number of cells counted manually and that recognized by our algorithm, suggesting that the results obtained from our automatic recognition algorithm was in good agreement with the manual counting results . The narrow bank of the confidence interval further suggests that the high correlation result is highly reproducible . One may note that the coefficient of the regression line was 0.445, meaning that the automatic recognition algorithm tends to count twice as many as that by the manual counting . The systematic overestimation of the algorithm could be due to the fact that the algorithm counted the ihc stains regardless of their size, whereas manual counting may have ignored small stains . A feasible approach to improve consistency is adding a size filter to the stain recognition algorithm . Nonetheless, this discrepancy does not invalidate our stain distribution images, since the high correlation coefficient ensured that our recognition algorithm revealed the same distribution pattern and only differed by a scaling factor . The regression analysis shows the correlation between the number of stain recognized by our stain recognition algorithm and that by the manual counting . The analysis has included 64 observations and the regression line is presented between the 95% confidence band . The regression analysis shows an r = 0.8029 and a correlation coefficient of 0.8961, suggesting a high correlation between manual counting and our stain recognition algorithm the in - vivo cellular mri is shown in figure 7, where the t2 * -weighted image (left) was compared with the ed1 stain distribution image (right). The animal from our rat heart transplantation model developed acute cardiac rejection and uspio was used as a macrophage - labeling agent in the cellular mri . As shown in figure 7a, the signal - void artifact in the t2 * -weighted mr image, as illustrated by the arrow, suggested the accumulation of uspio - labeled macrophages in the endocardium of the left ventricle . In comparison, our ed1 stain distribution image shown in figure 7b illustrated a similar pattern of accumulation of ed1 macrophages in the endocardium . One may note that the t2 * -weighted image does not match perfectly with the ed1 stain distribution image . This may be due to the fact that there could be tissue distortion during the fixation procedure and that the images may not align perfectly with the pathology slides . Despite this difference, the highly similar pattern suggested that our method can be used to reveal ed1 macrophages within pathology slides . The in - vivo cellular magnetic resonance (mr) images correlates with the ed1 stain distribution image . (a) the in - vivo t2 * -weighted cellular mr image, where the macrophages labeled with ultrasmall superparamagnetic iron - oxide particles create hypo - intensity patches (annotated by arrows) in the myocardium that develops acute allograft rejection . (b) the corresponding ed1 stain distribution image shows a similar pattern of the macrophages accumulation in the endocardium . The hypo - intensity patches in (a) correspond to the hyper - intensity locations in (b) the ex - vivo mrm is shown in figure 8, where the t2 * -weighted image of the ex - vivo tissue (left) was compared with the ed1 stain distribution image (right). The rat was from our model of ischemia / reperfusion injury, with mpio particles used as the macrophage labeling agent in the cellular mrm . The tissue section is outlined (black) in the ed1 stain distribution image to facilitate comparison . As shown in figure 8a, the mpio - labeled macrophages generate dark patches and spots in the mrm images, suggesting massive macrophage infiltration in the myocardium with ischemia / reperfusion injury . Similarly, our ed1 stain distribution image shown in figure 8b showed the same pattern of ed1 macrophage infiltration in the left ventricle . The high similarity of both imaging approaches confirmed that our method can reliably identify ed1 macrophages within pathology slides . The ex - vivo cellular magnetic resonance microscopy (mrm) correlates with the ed1 stain distribution image . (a) the ex - vivo t2 * -weighted cellular mrm, where the macrophages labeled with micron - sized iron - oxide particles create hypo - intensity patches in the myocardium that has ischemia / reperfusion injury (b) the corresponding ed1 stain distribution image shows highly similar pattern of macrophages accumulation in the myocardium, suggesting that the ed1 stain distribution image can reveal the macrophage infiltration across an entire pathology slide figure 9a shows the spatial distribution of the cell nucleus, lipofusin and red blood cell in 3 rats with heart ischemia / reperfusion injury . The stain recognition algorithm was trained to recognize cell nuclei (n), lipofusin (l) and red blood cells (r) under h and e stain, as shown in figure 9b . The distribution of cell nuclei can be used to investigate the increase of immune cells during the inflammation process, whereas the distribution of lipofusin may be used to investigate oxidative stress in the myocardium . Both three distribution images offer paranomic pathology information of the ischemia / reperfusion injury across tissue sections . The spatial distribution of cell nucleus, lipofusin and red blood cell in rat hearts with ischemia / reperfusion injury . (a) the spatial distribution of cell nucleus, lipofusin and red blood cell in 3 rats . (b) the stain recognition algorithm was trained to cell nuclei (n), lipofusin (l) and red blood cells (r) under h and e stain . These three distribution images offer paranomic pathology information that facilitates quantitative characterization in histopathology figure 10 is an example of feature distribution image showing the spatial distribution of the cell nuclear diameter . The diameter of the recognized cell nuclei was estimated using the kernel regression formulated in equation 2 . The feature distribution image can be used to provide a quantitative evaluation of a particular feature and to assist tissue characterization in histopathology . A feature distribution image showing the spatial distribution of the cell nuclear diameter . The cell nuclei were recognized under h and e stain and the spatial distribution of their diameter can be estimated using kernel regression . In this paper, we report on an automated method that conducts whole slide pattern recognition to obtain the stain distribution . Regression analysis showed a correlation coefficient of 0.8961 between manual counting and our stain recognition algorithm, suggesting that the labor intensive manual counting may be replaced by our automated recognition algorithm, which is critical for further analysis of the image data from wsi . Moreover, the ed1 stain distribution image was in good agreement with both in - vivo cellular mri and ex - vivo cellular mrm . The high density areas revealed in the ed1 stain distribution images correlated well with the hypo - intensity areas generated by iron - oxide - labeled macrophages, suggesting that our method can serve as a surrogate to reveal the distribution of macrophages across tissue sections . The novelty of our approach includes the following: first, we have implemented a parallel processing approach that makes whole slide pattern recognition possible . It is possible that the same processing approach can be combined with more advanced pattern recognition algorithms to extract meaningful features from pathology / ihc digital slides . Second, the stain distribution images obtained from our method offered a panorama of pathology information instead of a fraction of a view under the light microscope . This panoramic view is less susceptible to errors caused by subjective selection of the data . In contrast, traditional pathology validation often requires expert selection of the lesion site, making the process subjective and potentially biased . Whole slide pattern recognition leaves no bias and presents all the information across the pathology slide to offer more objective results . Third, the stain distribution images are quantitative information that can be further analyzed statistically . This unique feature is provided by the kernel density estimator, which is a widely - used regression approach in the field of statistics and has been used in a variety of real world applications . Lastly, our method can be fully automated and no human intervention is needed to obtain the stain distribution mapping . This offers a highly objective and efficient method that may enable a high throughput processing of data and massive screening . Despite the many advantages mentioned above, there are several possible pitfalls in our method that are noteworthy of discussion . The accuracy of our method depends on the performance of the recognition algorithm and it is possible that an algorithm may give false positive results secondary to poor slide preparation or artifacts in the preparation . Similarly, the stain variability may alter the recognition results and may create a false perception of the changes in the stain distribution . Both pitfalls may lead to misleading results that are hard to identify and thus, a post - processing inspection may be needed for quality control . First, we did only a qualitative comparison and did not conduct rigorous registration to compare our stain distribution imaging with mri . This limitation is due to the fact that the tissue specimen deforms substantially in the fixation process and the spatial information between mri and wsi cannot be perfectly aligned . Furthermore, the ex - vivo mri of the tissue - embedded paraffin block cannot be acquired because the mr spectrum is dominated by the paraffin resonance . Another limitation of our experiment is that we did not examine the positive and negative predicting values of our stain recognition algorithm . Although the accuracy of the morphology - based stain recognition has already been examined, it is unclear whether the same performance can be achieved in counting macrophages due to their highly pleomorphic shapes . The current setting is optimized to identify stains with an oval shape, but there can be other morphological presentations in the pathology slides . To overcome this limitation, the algorithm can be customized to accommodate different morphology of the stains . Moreover, although there is a positive correlation between the number of the recognized stains and manual cell counts, our stain recognition algorithm still tends to count more than that of manual counting . This may be due to the fact that macrophages have cytoplasmic projections that can lead to separated regions and result in duplicated counts . In addition, the nuclei of macrophages are often lobulated, further adding the complexity of the cell recognition in our use case scenario . Counting last, the stain distribution value can be affected by several factors, including the thickness of the sections, the z - depth and the bandwidth used in the kernel density estimator . Therefore, a comparison based on the stain - distribution value should be conducted in a carefully controlled manner . The obtained stain distribution image represents a panorama of the pathology information and can be used in both qualitative and quantitative analyses . This imaging approach may also assist pathologists in clinical diagnosis by offering them a toolkit for multiple applications in diagnostic pathology as well as yield newer methods to use image analysis to quantitate and analyze biomarkers, which are both diagnostic and prognostic . In addition, the algorithm described in this study may serve as a new and systematic approach to validate other imaging modalities . The ed1 stain distribution image confirms that the cellular mri technique is able to detect the macrophage infiltration of cardiac transplantation rejection or cardiac ischemic injury in our rat models.
Among the entities that cause dilated cardiomyopathy in children are dystrophinopathies, including duchenne muscular dystrophy (dmd), becker muscular dystrophy (bmd), and x - linked dilated cardiomyopathy, allelic conditions caused by defects in dystrophin (1). Dystrophin is an important cytoskeletal protein encoded by the dmd gene located at xp21.2 (2). Dmd is caused by complete loss of the dystrophin protein complicated by progressive skeletal muscle dystrophic pathology, whereas bmd often results from expression of an altered dystrophin protein leading to milder and a more variable clinical presentation than dmd (3). Approximately 70% of bmd patients develop dilated cardiomyopathy (dcm) mostly in the third decade of life or later (4, 5); they rarely develop severe dcm in childhood (6). Conserving the reading frame of the protein mutations in which the reading frame is conserved resulting in a functional amino- and carboxy - terminus with little effect on the central rod domain yield a milder phenotype consistent with bmd (7), whereas mutations that disrupt the reading frame cause a more severe phenotype consistent with dmd . The reading frame rule was found to be 9192% consistent in predicting phenotype in simplex cases of young boys (8). However, more recent studies have shown that this rule tends to be better at predicting phenotype for dmd, and there are more exceptions to this rule in bmd (9, 10). Genotype - phenotype correlations for specific mutations in dystrophin related to predisposition to or protection against dcm have been proposed (6, 11), but exactly how this leads to dcm is poorly understood . Here, we present a family in which two siblings and their nephew are affected by similar skeletal muscle abnormalities consistent with bmd . They have a novel frameshift mutation in the dmd gene responsible for this unique clinical phenotype of bmd and severe early - onset dcm . The importance of genetic screening of the male family members for early detection of lethal congestive heart failure (chf) will be discussed . There are three affected males in this family spanning two generations (figure 1a). The first affected member of the family was diagnosed with bmd at 6 years due to mild skeletal muscle weakness (ii-3). Dystrophin immunostaining in a muscle biopsy was consistent with bmd, and the dystrophin level was quantified as 310% of normal by western blot (athena diagnostics, worcester, ma). He was diagnosed with attention deficit hyperactivity disorder (adhd), but otherwise remained relatively symptom - free for a long time . At 12 years, however, he developed persistent cough resistant to conventional bronchodilator treatment . Initial echocardiogram revealed dilated lv with severely diminished lv systolic function (left ventricle shortening fraction of less than 10%). His clinical status continued to deteriorate despite maximum anti - congestive medications, and he was admitted for further management of intractable chf . Hemodynamic assessment by cardiac catheterization showed severe lv dysfunction: lv end - diastolic pressure (lvedp) 35 mmhg and cardiac index (ci) 1.8 l / min / m under mechanical ventilation and continuous intravenous inotrope infusion . He died at age 14 due to a cerebral thromboembolic event despite maximum medical treatment while awaiting a heart transplant . His younger brother (ii-4; 14 years) was diagnosed as bmd at 3 years of age with mild muscle weakness and failure to thrive . He has cognitive impairment with function in the range of 68 years old (at 14 years old) and a seizure disorder of unknown etiology . His cardiac status has been followed closely since 6 years of age because of his brother s history of early - onset of progressive dcm . Sequencing (emory genetics laboratory, decatur, ga) revealed a novel frameshift mutation in exon 27 of the dmd gene (c.3779_3785delctttggainsgg) in which 7 base pairs are deleted and two are inserted (figure 1b). This predicts an amino acid substitution and premature termination in the novel reading frame 8 codons downstream (p.thr1260argfs*8). No other potential disease - causing variants were found in the gene . Despite this predicted truncation of protein translation, immunofluorescence studies of a quadriceps muscle biopsy at age 14 instead showed reduced staining for dystrophin and other proteins of the dystrophin - glycoprotein complex and upregulation of utrophin consistent with bmd (figure 2). The greatest reduction in dystrophin immunofluorescence (perhaps absence of staining) was with an antibody that detects an epitope encoded by exons 27 - 28 (figure 2f). Dystrophin epitopes on either side of exons 27 - 28 were present (figure 2c, d, e, and g). Dystrophin was also detected in western blots from the same muscle biopsy using a carboxy terminus antibody . The total amount of the patient s dystrophin was greatly reduced, and the molecular weight appeared to be slightly reduced (data not shown). He had normal cardiac function through age 11 years, after which progressive lv systolic dysfunction and dilatation were noted by echocardiogram . His symptoms of chf continued to worsen over the next 2 years despite escalating medical treatment, and he was admitted to the hospital at 14 years because of severe respiratory distress and cardiac cachexia . Echocardiogram showed markedly dilated lv (lvidd 7.7 cm: z - score + 9.1) with severely diminished lv systolic function (lvsf 15%). Initial hemodynamic assessment by cardiac catheterization was consistent with mild to moderate lv dysfunction under continuous intravenous milrinone infusion (lvedp 12 mmhg and ci 2.1 l / min / m). His clinical condition deteriorated despite maximum medical treatment including mechanical ventilation, and eventually he underwent implantation of left ventricular assist device (heartware hvad, framingham, ma). He was discharged home with improved quality of life . The 7 year old nephew (iii-1) of these two affected boys he failed to thrive despite maximizing nutritional efforts (<1 percentile) and showed generalized muscle weakness and mild developmental delay . At 18 months he was noted to have serum ck above 13,000 iu / l, and he has been followed regularly since that time with a presumed diagnosis of bmd . Molecular analysis revealed identical familial frameshift mutation in dmd to his uncle (ii-4). At 18 months he was noted to have serum ck above 13,000 iu / l, and he has been followed regularly since that time with a presumed diagnosis of bmd . Female carriers in the family, the siblings mother (i-2) and their sister (ii-1), have had no symptoms of muscle weakness or chf . There is no further family history is available for i-2, as she was adopted in her early childhood . We report a family in which two male siblings with bmd developed severe, early - onset, progressive dcm . Both siblings developed intractable chf by age 12 that was resistant to conventional anti - congestive medications . Bmd patients are known to have milder skeletal muscle involvement than dmd patients and commonly develop dcm in their third decade or later . Cardiac dysfunction is a more frequent primary cause of death in bmd than in dmd (4, 12, 13). There are sporadic case reports of early onset dcm in bmd, but our patients are unique because of their earlier onset, multiple family member involvement with the same clinical phenotype, and a novel genotype . Dystrophinopathy refers to a clinical spectrum of mutation of dmd gene encompassing bmd, dmd, and x - linked dcm . X - linked dcm can present as a similar clinical course as ours, as reported by others (1416). However, all 3 patients here initially presented with primary skeletal muscle weakness with markedly increased serum ck, which is quite different from x - linked dcm where there should be no or very little skeletal muscle involvement (14, 15, 17). Skeletal muscle biopsy demonstrated that two brothers showed markedly reduced but positive dystrophin protein expression consistent with bmd . Collectively, we believe the affected brothers have bmd with severe early - onset dcm rather than x - linked dcm with coincidental skeletal myopathy . Disproportionally severe cardiac phenotype has been reported in limb - girdle type muscular dystrophy with relatively mild skeletal involvement (18). The x - linked dmd gene has 79 coding exons covering 2.6 million base pairs . The large size of the dmd gene makes it vulnerable to mutation, mostly by deletion of one or more exons . Dmd is caused by genetic mutations that lead to disruption of the reading frame of the dystrophin transcript and premature abortion of dystrophin synthesis (19). Bmd mutations usually do not disrupt the translational reading frame, and are typically in conjunction with either exon deletions or point mutations that alter splicing such that the resultant mrna maintains the open reading frame and leads to production of a partially functional but aberrant protein (2022). Previous reports described patients with bmd phenotype with nonsense mutations as a result of altered exon splicing and exon skipping, excluding the nonsense mutation containing exon (20, 23, 24) in our bmd patients (ii-4 and iii-1), we postulate that the mutation in exon 27 (c.3779_3785delctttggainsgg) results in altered splicing and mrna that allows production of a truncated dystrophin protein . This interpretation stems from immunohistochemical analysis of a muscle biopsy that demonstrates expression of exons proximal and distal to exon 27 (see figure 2). Unfortunately, we were unable to recover mrna of sufficient quality from archived muscle to test this hypothesis directly, but we note that disruption of the splicing enhancer sequence within exon 27 of the dmd gene by nonsense mutation can induce partial exon skipping, and thus results in a bmd phenotype (20, 21). However, this early - onset of severe progressive dcm in bmd patients, as seen in this family, has not been reported and cannot be solely explained by the exon skipping mechanism . Kaspar et al . Proposed that the deletions affecting the amino - terminal domain (exons 2 - 8) are associated with the early - onset of dcm (6), but it does not appear that our cases follow this rule . Double or multiple gene mutations are known to be responsible for more severe clinical phenotype in some hypertrophic cardimyopathy (hcm) (2527) and rarely in dcm (28). The possibility of second mutation in our cases cannot be ruled out, as we have not performed sequencing of other single gene causes of dcm or whole - exome sequencing . Genetic predictors of early onset of dcm in bmd patients have been studied extensively (6, 11), but the results are not conclusive as to which genotypes predict a worse natural course . There may be a fundamental difference in regulation of dmd gene transcription between skeletal and cardiac muscle . In this family, all three affected children developed a nonspecific central nervous system abnormality, but the clinical hallmark of the two brothers is early - onset lethal dcm . Because early detection of this progressive dcm is essential for optimum management, further effort in investigating genotype - phenotype correlation in bmd should be encouraged.
The prevalence of glaucoma, a leading cause of vision loss, increases with age . The number of people with glaucoma in australia is predicted to increase from 208,000 in 2005 to 379,000 in 2025 . The most common form of medical management of glaucoma is the use of topical eye drops that reduce intraocular pressure . -adrenergic antagonists (-blockers) are the most commonly prescribed glaucoma medicines in a number of countries including the united kingdom and the united states . In australia, treatment options include prostaglandin analogues, which are now the most common treatment for glaucoma . -blockers still have substantial usage and many combination products are available which contribute to their use . Timolol is a potent nonselective -blocker and was the mainstay of glaucoma therapy through the 1980s and 1990s [2, 7] because it is effective in lowering intraocular pressure, associated with few ocular side effects and does not affect pupil size [9, 10]. Although administered topically, when used in the eye timolol can reach the systemic circulation through the nasolacrimal duct, the conjunctival vessels, and gastrointestinal tract [1113]. A dose of one drop of 0.5% timolol solution to each eye is equivalent to a 10 mg oral dose [14, 15]. Systemic adrenergic -blocking effects of ophthalmic timolol may therefore occur, including effects on cardiac, pulmonary, central nervous system, and endocrine functions [6, 16]. Change in heart rate is one of the effects of the systemically absorbed fraction of ophthalmic timolol [6, 17]. In the first seven years of commercial production of ophthalmic timolol in the united states, 450 serious adverse cardiopulmonary events were reported, and 32 deaths were attributed to the use of ophthalmic timolol . Preexisting cardiovascular disease was reported in 31% of the 212 persons for whom medical history was provided . Ophthalmic timolol is therefore contraindicated in patients with certain cardiovascular disorders, including bradyarrhythmias and atrioventricular block . A number of studies have been conducted to confirm the systemic -blocking effects of ophthalmic timolol on cardiovascular functions, including bradycardia, and results varied substantially across studies . Randomised controlled trials and crossover studies found a range in reduction of resting heart rate and in peak heart rate during exercise, from negligible to an 11 beat per minute (bpm) reduction and from 5 to 22 bpm reduction, respectively, depending on the types of ophthalmic timolol: 0.250.5% aqueous or 0.10.5% hydrogel formulations [13, 1933]. Given these findings, we aimed to quantify the potential risks of hospitalisation for bradycardia following initiation of ophthalmic timolol in an elderly population . The australian government department of veterans' affairs (dva) administrative claims database was used in this study . Details of all prescription medicines, medical and allied health services, and hospitalisations for which dva pays a subsidy are available . Data are available for a treatment population that in september 2011 was 242,147 people and who had a median age of 80 years . Dva maintains a client file, which includes data on gender, date of birth, date of death, and family status . Medicines are coded in the dataset according to the world health organization (who) anatomical therapeutic chemical (atc) classification and the schedule of pharmaceutical benefits item codes . Hospitalisations are coded according to the international classification of diseases, version 10, australian modification (icd-10-am). The self - controlled case - series design [38, 39], which is a within person design, was used to compare the rate of hospitalisation for bradycardia during periods of exposure to timolol compared to unexposed periods . Eligible persons were those who were hospitalised for bradycardia (primary diagnosis icd-10am r001, i440, i441, i442, i443, and i495) between july 1, 2003, and june 30, 2009 . Persons were included if they were aged 65 years or over at the start of the study, eligible for all health services subsidised by dva and were dispensed at least one medicine in the year prior to the start of the study . To focus this analysis on new users of ophthalmic timolol, subjects who were dispensed ophthalmic timolol (atc code s01ed01) or combination medicines with timolol (atc code s01ed51) in the year prior to the start of the study were excluded . Subjects were followed until death or the end of the study period (june 30, 2009), whichever occurred first . As timolol eye drops are required to be discarded four weeks after opening, exposure duration for each dispensed ophthalmic timolol was defined as 35 days, allowing for an additional week to account for late prescription refill . The end of the exposure period was defined as 35 days after the last dispensing of timolol eye drops where no subsequent dispensing occurred . For those patients who had at least one timolol dispensing during the study period, their exposed time was partitioned into the following risk periods: 130 days, 31180 days, and all remaining exposure time after timolol initiation (> 180 days). A preexposure risk period of 30 days prior to initiating treatment with timolol was included to ensure the occurrence of the outcome was not altering the probability of subsequent exposure, a fundamental assumption of the self - controlled case series method . The actual day of prescription was excluded from this analysis as we were unable to define the temporal association between the exposure and a hospitalisation if they occurred on the same day . The incidence of outcomes in each of the exposure risk periods was compared to the incidence of outcomes in the unexposed reference period . Incidence rate ratios (irrs) were calculated using conditional poisson regression adjusting for age at hospitalisation and calendar year . Sensitivity analyses were performed by including additional adjustments for covariates including concomitant prescribing of oral beta - blockers (atc code c07), calcium - channel blockers (atc code c08), digitalis glycosides (atc code c01aa), and antiarrhythmics (atc code c01b). We also included patients who were hospitalised for bradycardia but not exposed to timolol during the study period to adjust for the possibility of increasing incidence of bradycardia hospitalisation with age . These patients contributed information on the impact of time varying covariates, including age, on the risk of the outcome [38, 39]. All person - time for the unexposed group was included in the unexposed reference period . All analyses were performed using sas version 9.12 (sas institute, cary, nc). There were 6,373 veterans with at least one hospitalisation for bradycardia during the study period, with 267 exposed to timolol and 6,106 never exposed . There was no statistically significant increase in the risk of hospitalisation for bradycardia in the first 30 days after initiating timolol (incidence rate ratio (irr) = 1.40; 95% confidence interval (ci) 0.872.26). The risk of bradycardia was significantly increased in the 31 to 180 days after timolol initiation (irr = 1.93; 95% ci 1.302.87), but did not remain statistically significantly elevated thereafter (table 2). Results were similar after adjusting for other conditions and when including unexposed patients (table 3). The stratified analyses also show similar risk estimates for patients not taking oral beta - blockers; however, the risk in the 31180-day risk period was not statistically significant (irr = 1.76; 95% ci 0.883.50) (table 3). Timolol is a nonselective -blocker; thus it is a risk factor for cardiovascular functions . Most of the published evidence of the systemic -blocking effects of ophthalmic timolol on cardiovascular functions was from randomised controlled trials (rcts), cross - over studies, or case reports [13, 1833]. Participants included in rcts were often very selective (e.g., healthy people) and not representative of the real - world population . Rcts excluded the elderly in whom -blockade has been found to be stronger and last longer . In addition, rcts were limited to investigating the impact of timolol on resting heart rate and peak heart rate during exercise; however, the impact of timolol on the more serious outcome of hospitalisation for bradycardia was not assessed . Using the dva administrative claims database in this observational study, we found an increased risk of hospitalisation for bradycardia one month after initiation of timolol eye drops . The increased risk of hospitalisation for bradycardia was reduced and no longer statistically significant after six months of continuous treatment . One explanation for this finding may be that those patients who continue to take ophthalmic timolol for extended periods are those with better tolerance, thus being less likely to experience the adverse event . Our findings are in line with previous clinical trial evidence and case reports which suggested that ophthalmic timolol was associated with adverse cardiac effects [18, 40]. Despite evidence linking the use of topical -blockers to bradycardia, codispensing of ophthalmic -blockers with medicines which can cause or exacerbate bradycardia is common . We previously showed that 36% of those with glaucoma who were dispensed verapamil were also codispensed ophthalmic timolol, a contraindication which may worsen bradycardia . The majority of glaucoma medicines are initiated by ophthalmologists, while adverse events may be managed by general practitioners, raising challenges of how to address adverse events across the continuum of care . Cross - specialty cooperation is therefore needed to optimise patient care with improved communication among ophthalmologists, general practitioners, pharmacists, and patients regarding the history of cardiac diseases and glaucoma treatment . The use of the self - controlled case series design where the patient implicitly acts as their own control adjusts for all confounders that remain fixed over the observation period, including sex, location, genetics, and underlying state of health . The absence of diagnostic information in the dva dataset means that disease severity could not be taken into account . However, sensitivity analyses were performed by adjusting for concurrent medications uses, which are the proxy for the presence of conditions that may impact on the risk of hospitalisation for bradycardia . These analyses made little difference to the risk estimates suggesting that the increased risk of hospitalisation for bradycardia is unlikely to be due to confounding because of changes in disease severity . In australia, ophthalmic timolol is registered for ocular hypertension and glaucoma, and the dva dataset does not allow distinguishing which condition the exposed individuals had . In addition, dosage information is not available in the dataset, so we were unable to assess the dose - response relationship . The selection of the veteran population in this study may be seen as another limitation for the generalisation of our findings . However, previous research has shown that there was no difference in use of practitioners, health services, and pharmaceuticals between war veterans and nonveteran patients in both the primary and tertiary australian care sectors after adjustment for age, service - related disability, and marital status . Our results, which have consolidated scientific evidence on the risk of hospitalisation for bradycardia, are therefore likely to be applicable to the elderly australian population and suggest that this adverse event is still occurring . Practitioners should be reminded to carefully examine the patient history before choosing a glaucoma regime and closely monitor patients after treatment initiation with topical nonselective beta - blocker eye drops to minimise adverse events and potentially avoid hospitalisations.
Thymoglobulin, rabbit anti - human thymocyte immunoglobulin (atg), is an immunosuppressive drug used as an anti - rejection therapy in solid organ transplantation and in hematological diseases . An association between atg and acute lung injury was first described in an experimental model in 1975 . Since then, few cases have been reported from which it is believed that in rare cases, atg is responsible for a spectrum of lung injuries varying from transient infiltrate to full - blown acute respiratory distress syndrome (ards). We report a case of atg - induced non - cardiogenic pulmonary edema (ncpe), its diagnosis and management under general anesthesia . A 28-year - old, 50 kg man with asa risk iii was posted for laparoscopic renal transplantation . He was diagnosed to have hypertension since 2 years and chronic interstitial nephritis leading to chronic renal failure (crf) since 6 months and was on maintenance hemodialysis twice a week . He had undergone laparoscopic cholecystectomy and bilateral nephrectomy 2 months back without any complications . In preoperative examination, his blood pressure was controlled with nifedipine 20 mg, metoprolol 50 mg, and clonidine 0.1 mg . Preoperative electrocardiogram (ecg) was normal nd 2-dimensionl echocardiography showed 60% ejection fraction with no wall motion abnormality . Induction was done with thiopentone sodium 350 mg, and suxamethonium 75 mg was given to facilitate endotracheal intubation . Right - sided internal jugular vein was cannulated by seldinger's technique for central venous pressure (cvp) monitoring . Continuous monitoring of ecg, pulse oxymetry, capnography, invasive blood pressure, cvp, airway pressure and temperature was done . For surgical procedure, airway pressure was 21 cm h2o after induction of anesthesia, which increased to 25 cm h2o after pneumoperitonium and stabilized at 28 cm h2o in head down position . After confirming negative reaction to test dose of atg and prophylactic administration of 100 mg iv hydrocortisone and 45.5 mg of iv chlorpheniramine maleate, 75 mg of atg diluted in 100 ml normal saline was started in central line which was to be given over a period of 4 hours . Methyl prednisolone 500 mg in 500 ml of normal saline was also started as an anti - rejection therapy . Intraoperatively, 3 l of normal saline and 100 ml albumin 20% was given to keep the cvp between 15 and 20 mm hg . Ureteric reimplant was done . At the end of surgery, which lasted for 4.5 hours, the urine output was around 2 l. throughout the procedure, all parameters remained normal, but at the time of port closure, suddenly airway pressures were elevated up to 3540 cm of h2o . On manual ventilation, resistance was felt . Gradually, oxygen saturation started falling to less than 90% even with 8 l / min of oxygen, with persistent high airway pressures . Cvp line was changed to pa catheter and pulmonary capillary wedge pressure (pcwp) of 15 mm hg was noted . Diagnosis of ncpe was confirmed . When surgery was completed, the patient was given inj . Diazepam 5 mg, furosemide 100 mg iv and shifted to icu . In icu, the patient was kept on ventilator on synchronized intermittent mandatory ventilation (pressure control)+ pressure support mode with 10 cm of positive end - expiratory pressure and 100% fio2) chest x - ray was done which showed diffuse infiltrates consistent with pulmonary edema . Gradually, as the patient's oxygenation and x - ray chest improved, he was weaned off the ventilator and extubated by next day morning . Pulmonary edema can be cardiogenic due to increase in the net hydrostatic pressure across the pulmonary capillaries or fluid overload or non - cardiogenic due to increase in the permeability of alveolar capillary membrane . Ncpe is characterized by simultaneous presence of severe hypoxemia, bilateral alveolar infiltrates on chest radiograph and no evidence of left atrial hypertension / congestive heart failure / fluid overload . The common causes are gastric aspiration, sepsis, trauma, and respiratory obstruction leading to negative pressure pulmonary edema . Less appreciated is the fact that various drugs, either taken as standard therapy or as an overdose, may precipitate ncpe . The first extensive review on agents causing pulmonary insult was published in 1972 which included 20 medications . Since that time bauman et al . Published a paper on drug - induced lung diseases in which atg is mentioned as a causal agent for drug - induced ncpe . Dean et al . And murdock reported atg - induced ards . Like the other case reports, it was impossible for us to definitely prove that pulmonary edema was secondary to atg . Cardiogenic pulmonary edema and fluid overload were ruled out by the absence of preexisting heart disease, good left ventricular systolic function and normal pcwp . The other causes of ncpe are less likely as it was an elective surgery, there was no respiratory obstruction and pulmonary edema developed at the end of surgery with endotracheal tube in situ . Pulmonary edema developed at the time of completion of atg infusion probably due to administration of antihistaminics and steroids during surgery . Although rechallenging can definitely establish the causal link, we did not believe it safe for purely diagnostic purposes . Diagnosing drug - induced ncpe is actually an exercise of exclusion as there is no diagnostic test available . It is related to the time proximity of administration of drugs, and pathogenesis involves direct cytotoxic insults to the lung epithelial cells and induction of cytokine triggered inflammatory responses . Various postulates are as follows: (1) atg contains antibodies of animal origin which are active against human t lymphocyte antigen, with cellular activation, degranulation and respiratory burst response damaging pulmonary endothelium . Pulmonary capillary endothelial permeability increases in response to tumor necrosis factor and alpha interleukin 1 and 8 released from damaged or activated lymphocytes similar to the pathogenesis of ards in sepsis . Our purpose of reporting this case is to alert anesthesiologists involved in transplantation program of this acute complication related to atg infusion so that they are prepared to treat it promptly.
In dental literature, cases of transmigrated mandibular canines are uncommon phenomenon.119 however some of them are far more extraordinary than others . Although transmigration of a tooth is out of the common, migration generally to apexes of molars is more uncommon event when compared with teeth which generally migrates to apixes of the incisor teeth . Besides, bilateral ones in vertical position may be more extreme than the others even though bilateral cases in horizontal position are extreme . Likewise, cases which reported in the past were consisted of only maxillary or mandibular canine teeth until camilleri3 discovered that multiple transmigration was also be more extreme than the others . However, we have aimed to present the more extreme cases of the transmigrated mandibular canines . Tk, 21 year - old girl, referred to our clinic complaining of a pain arising from her right mandibular permanent first molar . Panoramic radiograph revealed that horizontally impacted left mandibular permanent canine migrated crossing the midline located below the apices of the right mandibular second molar . The transmigrated canine located near the mandibular canal but there was no symptom associated with mandibular canal including; paresthesia, paralysis or pain . The migrated canine also showed no evidence of resorption or pericoronal radiographic changes suggesting cystic degeneration . Oral examination revealed that right mandibular canine was missing in the original arch and there was a swelling in the labial aspect of the symphsis . Panoramic radiograph revealed that right mandibular canine was impacted and crossing the midline under the apixes of the incisors and there was a compound odontoma in the original position of the right canine . A 28 year old woman turned to our clinic about the examination of her peridontal status . During the oral examination bilaterally overretained deciduous canines panoramic radiograph revealed bilaterally impacted canines lying vertically in the midline in the apices of the incisors . The radiographic appearance was in accordance with type 517 (figures 3 and 4). A 76 year old woman resorted to our clinic about the pain in right mandibular permanent first molar . The panoramic radiograph indicated that multiple teeth were impacted horizontally with part of the crowns crossing the midline . Occlusal radiograph delineated that three teeth were impacted including right mandibular permanent canine and two mandibular permanent incisors, and it confirmed transmigration of these teeth . Tk, 21 year - old girl, referred to our clinic complaining of a pain arising from her right mandibular permanent first molar . Panoramic radiograph revealed that horizontally impacted left mandibular permanent canine migrated crossing the midline located below the apices of the right mandibular second molar . The transmigrated canine located near the mandibular canal but there was no symptom associated with mandibular canal including; paresthesia, paralysis or pain . The migrated canine also showed no evidence of resorption or pericoronal radiographic changes suggesting cystic degeneration . A 16 year old girl had pain in the lower right third molar . Oral examination revealed that right mandibular canine was missing in the original arch and there was a swelling in the labial aspect of the symphsis . Panoramic radiograph revealed that right mandibular canine was impacted and crossing the midline under the apixes of the incisors and there was a compound odontoma in the original position of the right canine . A 28 year old woman turned to our clinic about the examination of her peridontal status . During the oral examination bilaterally overretained deciduous canines were observed and permanent canines were missing in the lower jaw clinically . Panoramic radiograph revealed bilaterally impacted canines lying vertically in the midline in the apices of the incisors . The radiographic appearance was in accordance with type 517 (figures 3 and 4). A 76 year old woman resorted to our clinic about the pain in right mandibular permanent first molar . The panoramic radiograph indicated that multiple teeth were impacted horizontally with part of the crowns crossing the midline . Occlusal radiograph delineated that three teeth were impacted including right mandibular permanent canine and two mandibular permanent incisors, and it confirmed transmigration of these teeth . This paper consists of some unusual cases of transmigration of the mandibular permanent canines such as, bilateral cases of the transmigrated canines in vertical position, one of the the most distant migration of canine from original side to contralateral to the apex of the third molar, and multiple transmigration case with transmigrated mandibular canine and impacted mandibular central incisors . The movement of a tooth is always in the direction of the crown, and most often the greatest amount of movement takes place prior to completion of tooth root development or at an earlier stage.8 ando et al15 reported that movement of transmigrated canine was more rapid before the formation of its root . Because of patient s age, it can be suggested that the greatest amount of movement occurred before completion of the root formation in case 1 . Rarely transmigrated canine was found under the first molar on the opposite side of the dental arch.1,6 auluck reported that presence of a canine migrated apical to the mesial aspect of right third molar.9 it was found that the transmigrated canine was lying horizontally under the second molar on the opposite side as shown in case 1 . The transmigrated canine in this case was one of the most migrated teeth concerning literature just as auluck s case . Joshi reported that 70.8% of the cases were overretained deciduous canines and 29.2% of the cases were exfoliated in the mandibular arch.1 this observation showed that resorption process of the root of the deciduous canine was rather slow in the absence of the developing permanent mandibular canine under the deciduous canine . In case 1, there are several reports related to transmigrated teeth pathologies including; a cyst or an odontoma,10,11 gardner s syndrome,12 or fracture.13 although nodine14 and ando et al15 reported that impacted and migrated mandibular canines were often discovered without producing any apparent symptoms and they did not observe any symptoms such as pain or oppression of mandibular nerve owing to the transmigration of canine in their patients, joshi1 reported that it was difficult to understand whether these pathologies were responsible for the transmigration process or the pathological situations occurred after the migration of the canine . In case 2, there was a compound odontoma which was associated with impaction or transmigration . In case 4 . Etiology was likely to be trauma, but we could not suggest any apparent reason of how or why canines had transmigrated in case 1 and in case 3 . Although peck20 reported that canine migration was congenitally inherited, none of closed relatives of the patients had this abnormality . A consensus on exact mechanism of transmigration has not been reached in literature yet . As a consequence camileri3 firstly reported multiple transmigration case including mandibular canine and lateral incisors . A patient with transmigrated mandibular right canine and impacted two lower incisors probably related to earlier trauma but, unfortunately, it is not sure whether these teeth were central or lateral incisor without surgery . According to camileri,3 impacted mandibular lower incisor teeth this case showed that eruption abnormalities might affect more than one type of tooth.16 while most of the transmigrated mandibular canines occurred unilaterally,1,2,4,5,7,9,11,17,19 only 9% of them were bilateral.1,6 although most of the bilateral transmigrated canines lied horizontally, there were no bilateral transmigrated teeth which were in vertical position . In case 3, while 33 lied vertically in the midline, 43 lied vertically between apices of the 41 and 42 because of inadequate space to cross . It cannot be suggested that both canines crossed the midline completely . In bilateral transmigration cases which canines lied in vertical position, it is impossible for both canines to cross the midline simultaneously . Transmigrated mandibular canines were classified according to mupparapu.17 it could be summarized as; type 1: the impacted canine is mesioangularly acrossing the midline, labial, or lingual to the anterior teeth with the crown portion of the tooth crossing the midline . Type 2: the canine is horizontally impacted near the inferior border of the mandible below the apices of the incisors . Type 3: the canine has erupted either mesial or distal to the opposite canine . Type 4: the canine is horizontally impacted near the inferior border of the mandible below the apices of either premolars or molars on the opposite side . Type 5: the canine is positioned vertically in the midline with the long axis of the tooth crossing the midline . Mupparapu s classification consisted of single transmigrated canine . In case 3, bilateral impacted canines in vertical position located in the midline was in accordance with type 5 . Besides the option of surgical removal, transplantation and surgical exposure with orthodontic alignment are treatment alternatives for the transmigrated teeth.2 a long term follow up without symptoms may be an alternative option for patients who are afraid of surgical processes . If it cannot be detected early, further treatment alternatives will lessen and the case might get worse . Surgical extraction is more appropriate than others in case of a pathological situation . Consequently, decision of treatment options relates to pathological conditions, location and position of the transmigrated tooth and patient s desire . In present study, only one of our 4 patients preferred surgical treatment (in case 2) and others went under follow up . The transmigrated canine, which was the subject of case 1, located near the mandibular canal . The report of brust18 clearly showed that the transmigrated canine maintained its nerve supply from the original site . Although the most probable treatment option is to keep the transmigrated teeth under critical observation with periodical panoramic examination, it is necessary to anesthetize the nerve bilaterally before the surgical extraction with local anesthesia in case of unexpected pathological condition . To conclude so far, some cases of them are rather extreme cases even though transmigration of mandibular canine is an unusual event . It is important to diagnose them in earlier stages of migration or abnormality to prevent more complicated situations . From the aspect of genetic factors, a lot of questions about etiology still remains unanswered, therefore detailed mechanisms of the transmigration origin is a subject of further genetic researches.
During august 2010december 2012, we recruited all patients who had clinically suspected m. ulcerans infection and had attended a clinic in the buruli ulcer endemic asante akim north district in ghana . Age- and sex - matched household contacts of patients were also asked to participate; all study participants were> 5 years of age . The study protocol was approved by the ethics review committee of the school of medical sciences, kwame nkrumah university of science and technology (chrpe/91/10). Whole blood samples were taken at baseline, at week 6, and at week 12 from 66 patients in whom the diagnosis of buruli ulcer disease had been confirmed by pcr for the is2404 repeat sequence specific for m. ulcerans (8); samples were also obtained from 20 household contacts at the same intervals . The samples were heparinized, and peripheral blood mononuclear cells were separated from 10-ml samples . Filarial infection was confirmed on a blood film stained with giemsa and delafield hematoxylin and examined for microfilariae at 10 and 40 magnification (the knott technique; 10). M. perstans nematodes were distinguished from l. loa and w. bancrofti nematodes by their small size and the absence of a sheath (figure 1). M. perstans nematodes can be distinguished from loa loa and wuchereria bancrofti nematodes by relative small size, detection in blood samples obtained during the day, and lack of a sheath . Patients in whom m. ulcerans infection was found were treated with 10 mg / kg oral rifampin and 15 mg / kg intramuscular streptomycin, administered daily at village health posts under direct observation for 8 weeks (rs8 treatment). The patients were followed up every 2 weeks in the clinic and monitored for complete healing or recurrence of skin lesions . We compared the proportion of household controls versus the proportion of buruli ulcer patients infected with m. perstans nematodes and the time to complete healing of m. ulcerans lesions in co - infected versus monoinfected patients . Categorical variables such as sex, clinical form of m. ulcerans lesion, and category of m. ulcerans lesion were compared by using the fisher exact test, and cumulative healing was compared by using the log - rank test . We found all forms of m. ulcerans disease among the group of patients; proportions of each type and category are shown in the table . Of 66 patients with m. ulcerans disease, 15 (22.7%) were co - infected with m. perstans nematodes, whereas 4 (13%) of 30 household controls had m. perstans infection (p = 0.4 by fisher exact test). Three patients in the co - infected group and none in the m. ulcerans monoinfected group reported pruritus . No other clinical signs of m. perstans infection were found . * na, not applicable . Comparison of combined m. ulcerans monoinfected and m. ulcerans co - infected with m. perstans versus household contacts, determined by 2-tailed fisher exact test . Comparison of m. ulcerans co - infected with m. perstans versus m. ulcerans monoinfected group, determined by 2-tailed fisher exact test . All 66 patients completed rs8 treatment, but 9 were lost to follow - up during the 12-month follow - up period . Buruli ulcer lesions healed completely in 14 co - infected patients by 58 weeks (median 20 weeks, 95% ci 14.630.2) and in 43 monoinfected patients by 50 weeks (median 21 weeks, 95% ci 16.725.5). We found no difference in cumulative time to healing for co - infected versus monoinfected patients (p>0.05 by log - rank test) (figure 2). Buruli ulcer patients who had m. perstans nematodes co - infection were treated with doxycycline (200 mg) and ivermectin (150 g / kg) daily for 6 weeks, starting during the second to fourth week of rs8 treatment . Viable microfilariae were still visible in peripheral blood mononuclear cell cultures from all co - infected patients after ivermectin and doxycycline treatment, but pruritus subsided in the 3 patients who had reported it . Survival analysis curve of cumulative healing for patients with mycobacterium ulcerans infection who were co - infected with mansonella perstans nematodes compared with those who had m. ulcerans monoinfection, ghana, august 2010december 2012 . No difference in cumulative healing was found between the 2 groups (p = 0.93 by log - rank test). We found co - infection with m. perstans in 23% of buruli ulcer patients in a disease - endemic district in ghana, but this prevalence was not significantly difference from prevalence among household contacts who served as controls (13%). As with buruli ulcer, m. perstans filariasis is predominantly found in rural populations and infection begins in childhood; the highest infection rates are found in children 1014 years of age (11), similar to those for children at highest risk for m. ulcerans infection . M. perstans infection occurs in ghana and was seen in the volta region of ghana around hohoe during the 1990s, but its prevalence is unknown (12), and no information is available about the average number of worms per infection . In uganda, prevalence of m. perstans infection has been found to range from 0.4% to 50% (13). M. perstans nematodes are transmitted by the bites of culicoides midges, but it is not known whether m. perstans infected midges can be co - infected with m. ulcerans . In a guinea pig model, skin penetration was shown to be a requirement for establishment of m. ulcerans disease (14), and it has been postulated that mosquito bites cause m. ulcerans disease in australia (6). These organisms might share a common route of transmission, but our findings in this small study do not support this concept . Our findings suggest that m. perstans nematodes are common in rural ghana and coincidentally infect patients with m. ulcerans disease, necessitating the consideration of these organisms in the management plan of buruli ulcer patients . Although often asymptomatic, m. perstans infection may cause eosinophilia, subcutaneous swellings, aches, pains, and skin rashes in a considerable proportion of patients (9). Because filarial nematodes are known to polarize the host immune responses from t - helper type 1 cells needed for protection against mycobacterial infections, toward humoral and t helper type-2 mediated immunity, we plan to undertake a study to investigate this interaction.
Despite ongoing advances in organ preservation and immunosuppression therapy, chronic liver allograft dysfunction (clad) remains the most common cause of patient morbidity and allograft loss in liver transplant patients . Liver allograft biopsy studies have shown that 37% of recipients who survive longer than 5 years present with clad, which adversely impacts the long - term allograft survival . The morphologic hallmarks of clad include hepatocyte necrosis, bile duct damage or disappearance, hepatic obliterative arteriopathy, and liver fibrosis [3, 4]. Liver graft fibrosis in particular is a major determinant of clinical outcomes in clad patients . These histopathologic changes associated with clad can be attributed to immunological and nonimmunological factors, including ischemia / reperfusion (i / r) injury, acute or chronic rejection, drug toxicity, and de novo or recurrent disease [6, 7]. Development of novel strategies that prevent clad - associated damage is the key to ultimate graft survival . Emerging evidence has indicated that chemokines and their receptors are associated with the development of clad [2, 8]. Cxcl4 is secreted by platelets that specifically activate the cxcr3 receptor, which is involved in the control of many biological processes, including hematopoiesis, angiogenesis, fibrogenesis, and innate and acquired immune responses . Cxcl4 expression has been observed in liver allografts throughout all stages of transplantation, indicating that cxcl4 and its receptor cxcr3 have important roles in the pathogenesis of clad after liver transplantation . However, its role in the pathogenesis of clad has not been completely elucidated . In this study, we applied isobaric tags for relative and absolute quantification (itraq) proteomics analysis to identify that cxcl4 is an informative gene in clad . We showed that the severity of clad was significantly ameliorated after cxcl4 neutralization by monoclonal antibody (cxcl4mab) treatment in a rat model of clad . Cxcl4 serum concentrations were determined in 93 liver transplant patients with clad and 20 healthy subjects . After histopathological evaluation, we extracted total hepatic mrna from paraffin - embedded liver biopsies of the patients with clad and 30 patients without clad . Cxcl4 serum concentrations were determined by enzyme - linked immunosorbent assay (elisa; r&d systems, mn). Total mrna from patients with and without clad was reverse - transcribed using superscript (invitrogen). Quantitative reverse transcription polymerase chain reaction was carried out for cxcl4 with an assay from applied biosystems (hs00236998_m1). Pathogen - free, healthy male bn (rt) and lewis (rt) rats weighing 250300 g (purchased from the institute of laboratory animal science, chinese academy of medical sciences) were used as liver donors and recipients, respectively . The animals were housed under standard conditions with 12-hour dark - light cycle and free access to water and food . Animal experiments were performed with the authorization of the animal care and use committee of the tianjin medical university . All surgical procedures were performed under aseptic conditions by a single microsurgeon with the aid of a 440x magnification microscope (leica microsystems, wetzlar, germany). The two - cuff technique was used to establish rat orthotopic liver transplantations (olt) models as previously described by kamada and calne . Daily cefazolin (40 mg / kg) was injected intramuscularly after the implantation operation for 5 days to prevent infection . Tacrolimus was donated by the organ transplantation center of tianjin first center hospital (tianjin, china) and administered (0.1 mg / kg per day, injected intramuscularly) for 5 days after the implantation operation to induce chronic liver allograft dysfunction . Group a (n = 36) included bn rat to lewis rat transplantation with a 30-day course of low - dose subcutaneous tacrolimus (0.1 mg / kg / day) treatment . Group b included control isogenic liver transplantations from bn rats to bn rats (n = 36) along with 30 days of low - dose subcutaneous tacrolimus (0.1 mg / kg / day) treatment . In group c (n = 20), liver transplantation was performed from bn rats to lewis rats with no immunosuppressive treatment . Each recipient rat was examined twice daily . For the survival experiment, recipient rats with clad were given either cxcl4mab (1 mg / kg) or physiological saline (ps) through the tail vein once every week from postoperative day (pod) 5 for 2 months . Antibodies against cxcl4 (sc-50300), cxcr3 (sc-9902), egfr (sc-373746), jak2 (sc-390539), stat3 (sc-8019), collagen iv (sc-167528), and gapdh (sc-48166) were from santa cruz biotechnology . The liver allografts were harvested for masson staining, immunohistochemistry, western blotting, and hepatic stellate cells (hsc) isolation . Serum biochemistry analysis included aspartate aminotransferase (ast) and total bilirubin (tbil) assessed by standard spectrophotometric methods using a hitac7170a automatic analyzer (hitachi, tokyo, japan). Statistically significant differences between groups were determined by one - way anova (p <0.01; p <0.05). Paraffin - embedded liver tissue sections of recipient rats were stained with hematoxylin and eosin (h&e). Liver histopathological analysis was performed by a transplant pathologist who was blinded to the treatment group assignment . Independent sample t - tests were used for statistical analyses with p <0.05 defined as statistically significant . Extracted proteins were assayed using the bca method and digested according to the fasp method . Itraq quantitative proteomic analysis was performed on triplicate samples of recipient rats liver allografts and on three independent liver allografts as described [13, 15]. After acquiring ms / ms data with q - exactive software (thermo - fisher scientific), all data files were processed using the proteome discoverer 1.3 software (ab sciex, foster city, ca 94404, usa) for peptide identification . The user - defined search parameters were enzyme: trypsin; static modification: carboxyamidomethylation (57.021 da); dynamic modification: m oxidation (15.995 da) and carbamidomethyl (c), itraq8plex (n - term), and itraq8plex (k); false discovery rate (fdr) of all peptide and protein identifications: <1%; max missed cleavages: 2; precursor ion mass tolerance: 15 ppm; fragment ion mass tolerance: 20 mmu . Significant differences between the experimental and control groups were determined by anova followed by tukey's multiple range test . A difference 1.50-fold was considered evidence of upregulation, whereas a difference of 0.67-fold was considered to indicate downregulation . Protein expression levels were considered statistically significant if the p value was <0.05 . To explore the pathogenesis of clad, gene ontology (go) ingenuity pathway analysis (ipa, ingenuity systems, http://www.ingenuity.com/) used to investigated the functional relationships of discriminated proteins . We used ipa and its accompanying interaction database to analyze the data in the context of molecular mechanisms and relate these molecular changes with organism physiology and pathophysiology and to identify the ranked enrichment networks and canonical pathways for discriminated proteins . Fisher's exact test was used to determine the probability that each biological function assigned to that data set was due to chance alone . Hscs were isolated from the liver allografts by a modified method as previously described [16, 17]. Briefly, liver grafts were perfused through the superior vena cava with 0.044% collagenase (sigma, st . Suspensions of liberated hscs were prepared by centrifugation on a double - layered (17%/11.5%) metrizamide solution (sigma, st . After centrifugation at 20,000 rpm for 15 min, the hscs were harvested from the top of the upper layer . Hsc preparations with greater than 90% purity and viability were routinely obtained using this procedure, as determined by ultraviolet - excited fluorescence microscopy and trypan blue dye exclusion, respectively . The shrna target sequence for cxcl4 was 5-cgctgaagaatgggagcaaaactcgagttttg ctcccattcttcagcg-3. A scrambled fragment (5-agcgacggagatctt agctgtctcgagacagctaagatctccgtcgct-3) was used as a negative control . Dna oligonucleotides to produce the plasmid - based shrna were cloned into the gv118 vector using the hpai / xhoi restriction sites . The lentiviral expression vector (gv118) and packaging vectors (pgc - lv, phelper1.0 and 2.0) were cotransfected into 293 t cells with lipofectamine 2000 according to the manufacturer's instructions . The supernatant was collected 48 h later, centrifuged (4000 g, 4c, 10 min) to remove cell debris, filtered through 0.45-m cellulose acetate filters, and then concentrated again (4000 g, 4c, 15 min). The lentiviral vectors expressed green fluorescence protein (gfp), which allowed for titering and measurement of their infection efficiency in transduced cells . Isolated hscs were dispensed into 6-well plates at a density of 50,000 cells per well and transduced with shrna - expressing lentivirus at a multiplicity of infection (moi) of 75 . Immunohistochemistry was performed for cxcl4 protein expression in five sections from per graft after the samples were fixed in 10% neutral - buffered formalin embedded in paraffin . The liver sections were incubated with a 1: 100 dilution of anti - rabbit monoclonal cxcl4 antibody (abcam, ab129183). Liver allograft samples were rapidly ground in liquid nitrogen, and the cultured hscs were harvested . The tissue powder or the isolated hsc cells were reconstituted in ice - cold ripa buffer with protease and phosphatase inhibitors . Supernatants were recovered after centrifugation and assayed for protein content using the bca method (beyotime biotechnology, p0010). Protein samples of 50 mg per lane were separated by 8% sds - polyacrylamide gel electrophoresis (sds - page) and transferred to polyvinylidene - difluoride (pvdf) membranes . The membranes were incubated in 5% skimmed milk for 2 h at 37c and overnight at 4c with primary antibodies (santa cruz) (rat anti - cxcr3, 1: 200 dilution; egfr, 1: 200 dilution; jak2, 1: 200 dilution; stat3, 1: 200 dilution; collagen iv, 1: 200 dilution). The membranes were exposed to the negative films to develop target bands after incubation with enhanced chemiluminescence (santa cruz, usa). The intensities of bands were quantitated by labworks 4.5 software (uvp, usa). The serum concentration of cxcl4 was significantly increased in patients with clad compared to healthy controls (mean 66.70 2.3 ng / ml, p = 0.026; figure 1(a)). We next validated whether hepatic cxcl4 mrna expression was also increased in liver transplant recipients . Indeed, cxcl4 mrna was significantly increased in subjects with clad versus individuals without clad (p = 0.019; figure 1(b)). These results suggest that the upregulated cxcl4 is particularly associated with clad in liver transplant patients . Low - dose tacrolimus (0.1 mg / kg / day) treatment after liver transplantation was administered to recipient rats in the bn - to - lewis transplantation group (group a). Survival in group a (recipient rats with clad) was 62.8 12.6 days . This survival was greater in group b (the low - dose tacrolimus - treated bn - to - bn group) (105.1 19.3 days, p <0.001) than in group a (figures 2(a), 2(b), and 2(c)). However, all recipient rats in group c (bn - to - lewis without tacrolimus treatment group) developed irreversible acute rejection and succumbed to disease within 20 days . Liver allograft function was evaluated by determination of serum ast and tbil levels . In group a, all liver grafts had moderate to severe dysfunction detected on pod 60 (ast 441.6 101.6 mg / dl; tbil 236.5 91.6 mg / dl) but no apparent alterations were observed in the control group (group b) (ast 62.76 15.6 mg / dl, p <0.001; tbil 43.3 10.2 mg / dl, p <0.001) (figures 2(a), 2(b), and 2(c)). Manifestations of clad in this study, namely, obliterative arteriopathy, bile duct dystrophy or loss, and liver fibrosis, were consistent with the diagnostic criteria proposed for clad . The architecture of the clad livers had changed from a lobular structure to a tissue largely composed of fibrotic nodules connecting portal tracts detected on pod 60 . Obliterative arteriopathy and bile duct dystrophy or loss were more evident in clad liver allografts (figures 2(a), 2(b), and 2(c)). However, no apparent histologic alterations and liver dysfunction were observed in the control group in which all rats survived> 90 days . In this study, we investigated clad - associated proteins in liver allografts from the clad (bn - to - lewis with tacrolimus) and control groups (bn - to - bn with tacrolimus). After validating the purity of the isolated proteins, biological groups containing proteins from the bn - to - bn group (113 or 115) and the bn - to - lewis group were labeled with itraq reagents (114 or 116) and then analyzed by nano - lc - ms / ms . Therefore, we set our acceptance criteria to include proteins with at least two unique labeled peptides . We then identified a total of 225 rat liver clad - associated proteins after combining four replicates . Of these, 98 proteins were reported previously, indicating the accuracy of the ld purification and proteomics techniques . All identified proteins (225) were categorized into 17 groups according to their function and their assigned subcellular locations based on the patrhe and go term websites (figures 3(a), 3(b), and 3(c)). The most unexpected findings were the identification of platelet - derived chemokines, epidermal growth factor receptor (egfr), and the jak / stat signaling pathway in the pathogenesis of clad (figures 3(a), 3(b), and 3(c)). Examining these groups, the platelet - derived chemokine cxcl4, jak / stat, and egfr signaling pathways were significantly represented (20.9%, 47 proteins). Another major protein group was involved in the integrin signaling pathway (approximately 8.9%, 20 proteins), including several bidirectional signal transmitters for focal adhesion formation and fibroblast activation . This finding raised the possibility that cxcl4 expression, fibroblast activation, and fibrogenesis may be involved in clad . Cytoskeletal regulation by rho gtpase (14, 6.2%), glutamine glutamate conversion (14, 6.2%), fructose galactose metabolism (14, 6.2%), vitamin d metabolism (13, 5.8%), asparagine and aspartate biosynthesis (14, 6.2%), and glycolysis (14, 6.2%) were all notable liver metabolism proteins identified from clad recipient rat livers . Another notable group was composed of 18 proteins (approximately 8% of the total identified proteins) including the signaling proteins implicated in t cell and b cell activation . We also identified other cell signaling proteins including interleukin signaling (10, 4.4%), g - protein signaling (9, 4.0%), and androgen / estrogen / progesterone biosynthesis (2, 0.9%) in this study . Finally, there were a substantial number of mrna splicing proteins (15; 6.7% of the total) not belonging to the other 16 categories, which indicated that some functions of clad - associated proteins are still unknown and will require further study . By pathway and biological function analysis, six differential proteins involved in clad or liver graft fibrosis, including cxcl4, cxcr3, egfr, jak2, stat3, and collagen iv, were selected for validation . Immunohistochemistry analysis showed that significantly greater expression of cxcl4 at pod 60 was observed in all liver allografts in the clad group (bn - to - lewis) compared with those of the control group (bn - to - bn) (shown in figure 4). When validated by western blot analysis at pod 60, all six proteins were significantly overrepresented in all liver grafts in the clad group, compared with the bn - to - bn control group (shown in figure 4). Taken together, these findings indicated cxcl4 and its receptor cxcr3, the egfr signaling pathway, the jak2/stat3 signaling pathway, and collagen proteins participate in the pathogenesis of clad . These signaling pathways have potential therapeutic roles for liver fibrosis induced by hsc activation and clad after liver transplantation . The activation of hscs is a key event in the pathogenesis of clad . To understand the molecular mechanisms underlying the induction of cxcl4 expression in clad, the isolated hscs were transfected with cxcl4 shrna for 72 hours and examined for fibrogenesis by western blot ., we found that cxcl4 shrna significantly reduced cxcr3, egfr, jak2, stat3, and collagen iv expression in the transfected hscs, but not in the control hscs (shown in figure 5) (p <0.01). These data suggested that activation of hscs is at least partially due to the induction of cxcl4 . This result also suggests that cxcl4 is upstream of cxcr3, egfr, jak2, and stat3 in the hsc activation pathways affected by clad . Considering the important role of cxcl4 in recipient rat livers after transplantation, low - dose tacrolimus - induced bn - to - lewis rat clad models were established in this study . We used a neutralizing monoclonal antibody (cxcl4mab) to block cxcl4 and to evaluate its protective roles in the pathogenesis of clad . For the blockade experiment, either cxcl4mab (1 mg / kg) or physiological saline were delivered to recipient rats (bn - to - lewis) through the tail vein once every week for 2 months beginning on pod 5 . The protective effect of the antibody was observed through serum biochemistry analysis, survival time after transplantation, and liver histopathological analysis . Importantly, cxcl4mab treatment alleviated recipient rat liver dysfunction after transplantation when compared with the physiological saline group (shown in figure 6). Survival time was more prolonged among recipient rats in the cxcl4mab - treated group than in the physiological saline group (p <0.001) (shown in figure 6(a)). The ast levels in the cxcl4mab - treated group were significantly lower (105.1 20.3 mg / dl) than in the physiological saline group (468.6 133.5 mg / dl, p <0.001). Similar trends were observed regarding tbil levels between the cxcl4mab - treated group (71.5 19.7 mg / dl) and the physiological saline group (260.7 84.6 mg / dl, p <0.001) (shown in figure 6(b)). Liver fibrosis in the recipient rat clad grafts was detected at pod 60 by masson stain (showed in figures 6(c) and 6(d)). We found that cxcl4mab alleviates recipient rat liver fibrosis after transplantation when compared with rats in the physiological saline group . Previous studies have demonstrated that cxcl4 is involved in hematopoiesis, angiostasis, organ fibrogenesis, mitogenesis, tumor growth, and metastasis . However, its role in the pathogenesis of clad has not been elucidated . In our study, to better understand clad, we analyzed rat liver transplantation samples with itraq proteomics analysis and nano - lc - ms / ms . These proteins were categorized into 17 groups according to their function and their assigned subcellular locations based on patrhe and go term analysis . We first identified cxcl4, cxcr3, egfr, jak2, stat3, and collagen iv as associated with clad pathogenesis and validated the relationship between cxcl4 and liver fibrogenesis in isolated hscs . Indeed, liver graft fibrosis, a hallmark of clad, is a major cause of morbidity and mortality after liver transplantation . We found that the protein expression of cxcl4 and its receptor cxcr3 were both upregulated in clad liver grafts . Cxcl4 is secreted from platelets that specifically activate the cxcr3 receptor, which forms the basis for their pertinent involvement in the pathogenesis of clad . A previous study showed that cxcl4 promotes fibrosis by platelet activation and aggregation, which activates or attracts hepatic stellate cells . Cxcl4(/) mice showed a significant reduction in histological and biochemical liver damage, which was accompanied by changes in the expression of fibrosis - related genes (tgf - beta [transforming growth factor beta], mmp9 [matrix metalloproteinase 9], and timp-1 [tissue inhibitor of matrix metalloproteinase 1]). More interesting, our findings also showed that clad was significantly ameliorated after blockade of cxcl4 in rat models (figures 6(a)6(d)). The contribution of the cxcl4-cxcr3 axis to liver fibrosis has been delineated in recent years . Cxcr3 is expressed on fibroblasts, epithelial and endothelial cells, smooth muscle, activated t lymphocytes, and dendritic cells . These cells express either cxcr3a, its splice variant cxcr3b, or a balanced combination of both . Stimulation of the cxcl4-cxcr3 axis leads to increased proliferation and activation of hscs, which are not only a target but also a source of chemokines that contribute to the liver fibrogenesis . To identify the signals downstream of cxcl4, cxcl4 shrna was used to transfect hscs isolated from clad liver grafts in this study . We found that cxcl4 shrna significantly reduced the expression of downstream fibrosis - related proteins (cxcr3, egfr, jak2, stat3, and collagen iv) in hscs (figure 5). Egfr, a transmembrane receptor tyrosine kinase, has been shown to play a key role during liver fibrosis following acute and chronic liver damage . Egfr is expressed on hscs, which are thought to be the major cell type involved in liver fibrosis . Studies have shown that the egfr inhibitor erlotinib prevented fibrosis progression by reducing egfr phosphorylation in hscs and lowering the total number of activated hscs [24, 25]. Although these results suggest that egfr inhibition might be a new therapeutic approach during liver fibrosis, the roles of egfr and its ligands in clad after transplantation are not completely understood thus far . Conditional deletion of cxcl4 (cxcl4 shrna) significantly reduced expression of fibrogenesis - associated proteins (cxcr3, egfr, jak2, stat3, and collagen iv) in hscs (figure 5). These data suggest that cxcl4 is upstream of cxcr3, egfr, jak2, and stat3 in hsc activation and liver graft fibrosis during clad . More interestingly, the significantly protective effects of cxcl4mab treatment in recipient rat liver dysfunction were observed in this study through serum biochemistry analysis, survival time after transplantation, and liver histopathological analysis . Taken together, these results indicated that the cxcl4-cxcr3 axis participates in the pathogenesis of clad and that cxcl4mab treatment is potentially beneficial in preventing and treatment of clad after liver transplantation . In conclusion, our study provides the first dataset regarding clad liver allograft proteins and their variants under pathological conditions . Cxcl4, cxcr3, egfr, jak2, stat3, and collagen iv were first identified as participating in the pathogenesis of clad in this study . Therefore, our results add new insight into the mechanisms of clad after liver transplantation . Therapeutic approaches that neutralize cxcl4, the newly identified target of fibrosis, may represent a novel strategy for preventing and treating clad after liver transplantation.
Between november 2001 and july 2008, nineteen patients (twenty feet) underwent modified weil osteotomy for freiberg's disease . We included patients who complained of persistent pain in spite of more than 6 months of conservative treatment at any stage according to smillie's classification system . Diagnosis was based upon clinical history, physical examination, and plain radiographs for all patients . Informed consent was waived because of the retrospective nature of the study . Under spinal anesthesia a pneumatic tourniquet was applied to the ipsilateral thigh and inflated to a pressure of 320 mmhg . Via the dorsal longitudinal approach, the extensor digitorum longus tendon of the affected toe was retracted laterally, exposing the mtp joint . In the beginning, the joint was debrided and a synovectomy was carried out before osteotomy . If a loose body, peripheral osteophyte, periarticular spur or synovitis were observed, they were removed or a synovectomy was performed . Afterwards, modified weil osteotomy was done via the distal metaphysis . In the dorsal aspect, most of the damaged area of the metatarsal head and neck area was removed in the bone wedge . Healthy plantar cartilage was rotated to the center of the joint for forming a new articular surface . Finally, the osteotomy was stabilized using a single screw (spin screw; integra lifescience co., plainsboro, nj, usa) with a low profile head and non threaded lag to the dorsal aspect of metatarsal neck (fig . Postoperatively, patients were allowed to bear weight as tolerated on their heel in an open, hard - soled surgical shoe . Upon radiographic evidence of healing at the osteotomy site, transfer of weight to the forefoot in a regular shoe the patients underwent periodic clinical and radiographic follow - up at 4 weeks, 3, 6, 12 months, and then annually . Relief of pain was evaluated using visual analogue scale (vas)19) rating from 0 to 10 . The patients were examined using a standardized questionnaire based on the american orthopaedic foot and ankle society (aofas) lesser metatarsophalangeal - interphalangeal scale.20) this score includes clinical variables such as pain, restriction of footwear, painful callus, functional restriction of the mtp joint, rom of mtp joint and alignment of the toes . In all cases, passive mobility of mtp joint the patient seated, the examiner stabilized the metatarsal with one hand while grasping proximal phalanx with the other hand . The examiner moved the toe cephalad and caudally in range of maximal area to assess dorsiflexion and plantarflexion . The patients' subjective satisfaction was also evaluated after surgery at last follow - up . Patients were asked to rate their result as excellent, good, fair or dissatisfied.21) although the smillie's classification system is based upon inspection of the metatarsal head, several features may be interpreted from plain radiographs.2,17) we reviewed preoperative weight - bearing radiographs and intraoperative finding to classify the stage of freiberg's disease according to the smillie's classification system (fig . We divided the patients again into early stage and late stage based on smillie's classification system.11,12,17,22) the early stage included smillie stage i to iii and the late stage included the rest, stage iv and v. in this study, dorsal closing wedge osteotomy was performed in both early and late stage of freiberg's disease . In preoperative radiograph, radiograph, metatarsal shortening due to surgery was analyzed by the modification of jones et al.23) in periodic radiographic follow - up, if bridging trabeculae across the osteotomy site emerged, it was considered as radiographic union . Other complications were also reviewed preoperatively and postoperatively such as callus, floating toe, stiff toe, transfer metatarsalgia, fixation failure, fracture of metatarsal, subluxation of the mtp joint, soft tissue irritation, nerve injury and nonunion or delayed union . Wilcoxon signed - rank test and mann - whitney u - test were performed to test the hypotheses regarding effectiveness of the procedure . Null hypotheses of no difference were rejected if p - values were less than 0.05 . Between november 2001 and july 2008, nineteen patients (twenty feet) underwent modified weil osteotomy for freiberg's disease . We included patients who complained of persistent pain in spite of more than 6 months of conservative treatment at any stage according to smillie's classification system . Diagnosis was based upon clinical history, physical examination, and plain radiographs for all patients . A pneumatic tourniquet was applied to the ipsilateral thigh and inflated to a pressure of 320 mmhg . Via the dorsal longitudinal approach, the extensor digitorum longus tendon of the affected toe was retracted laterally, exposing the mtp joint . In the beginning, the joint was debrided and a synovectomy was carried out before osteotomy . If a loose body, peripheral osteophyte, periarticular spur or synovitis were observed, they were removed or a synovectomy was performed . Afterwards, modified weil osteotomy was done via the distal metaphysis . In the dorsal aspect, most of the damaged area of the metatarsal head and neck area was removed in the bone wedge . Healthy plantar cartilage was rotated to the center of the joint for forming a new articular surface . Finally, the osteotomy was stabilized using a single screw (spin screw; integra lifescience co., plainsboro, nj, usa) with a low profile head and non threaded lag to the dorsal aspect of metatarsal neck (fig . Postoperatively, patients were allowed to bear weight as tolerated on their heel in an open, hard - soled surgical shoe . Upon radiographic evidence of healing at the osteotomy site, transfer of weight to the forefoot in a regular shoe the patients underwent periodic clinical and radiographic follow - up at 4 weeks, 3, 6, 12 months, and then annually . Relief of pain was evaluated using visual analogue scale (vas)19) rating from 0 to 10 . The patients were examined using a standardized questionnaire based on the american orthopaedic foot and ankle society (aofas) lesser metatarsophalangeal - interphalangeal scale.20) this score includes clinical variables such as pain, restriction of footwear, painful callus, functional restriction of the mtp joint, rom of mtp joint and alignment of the toes . In all cases, passive mobility of mtp joint the patient seated, the examiner stabilized the metatarsal with one hand while grasping proximal phalanx with the other hand . The examiner moved the toe cephalad and caudally in range of maximal area to assess dorsiflexion and plantarflexion . The patients' subjective satisfaction was also evaluated after surgery at last follow - up . Patients were asked to rate their result as excellent, good, fair or dissatisfied.21) although the smillie's classification system is based upon inspection of the metatarsal head, several features may be interpreted from plain radiographs.2,17) we reviewed preoperative weight - bearing radiographs and intraoperative finding to classify the stage of freiberg's disease according to the smillie's classification system (fig . We divided the patients again into early stage and late stage based on smillie's classification system.11,12,17,22) the early stage included smillie stage i to iii and the late stage included the rest, stage iv and v. in this study, dorsal closing wedge osteotomy was performed in both early and late stage of freiberg's disease . In preoperative radiograph, radiograph, metatarsal shortening due to surgery was analyzed by the modification of jones et al.23) in periodic radiographic follow - up, if bridging trabeculae across the osteotomy site emerged, it was considered as radiographic union . Other complications were also reviewed preoperatively and postoperatively such as callus, floating toe, stiff toe, transfer metatarsalgia, fixation failure, fracture of metatarsal, subluxation of the mtp joint, soft tissue irritation, nerve injury and nonunion or delayed union . Wilcoxon signed - rank test and mann - whitney u - test were performed to test the hypotheses regarding effectiveness of the procedure . Null hypotheses of no difference were rejected if p - values were less than 0.05 . The average age of the patients was 33.6 years (range, 17 to 62 years), and the mean follow - up period was 72.6 months (range, 41 to 121 months) (table 1). The aofas score increased significantly after surgery from 63.3 14.9 to 80.4 5.6 (p <0.0001). Vas improved from 6.2 1.4 to 1.4 1.5 at last follow - up (p <0.0001), and rom of the mtp joint increased from 31.3 10.1 to 48.3 13.0 degrees at last follow - up (p <0.0001). All patients were satisfied, reporting excellent or good results except one patient who had joint stiffness . The patient had rom limitation of 2nd mtp joint preoperatively and complained about no improvement of rom after surgery, not being satisfied with the surgery . On plain radiographs, joint space widening and degeneration of the mtp joint four cases were classified as smillie stage i, eleven cases as stage ii, one case as stage iii, two cases as stage iv, and two cases as stage v. there was no significant difference in variables such as improvement of vas, aofas score and rom of mtp joint after modified weil osteotomy comparing early stages with late stages (fig . 3). Initially, 14 cases had a shorter metatarsal compared to opposite side and the average of initial metatarsal shortening was 1.0 1.0 mm (range, 0 to 3.3 mm). Postoperatively, the mean metatarsal shortening due to operation was 3.4 1.7 mm (range, 0.9 to 5.8 mm) without including initial shortening . Radiographic union was achieved at 8.2 2.5 weeks (range, 4 to 12 weeks) after the osteotomy . Postoperative complications included callus on the plantar area of the third metatarsal head in three cases (15%), floating toe in one case (5%) and stiff toe in one case (5%). One patient complained about transfer metatarsalgia with callus on the plantar area of the third metatarsal head (5%). There was no evidence of fixation failure, fracture of metatarsal, subluxation of the mtp joint, soft tissue irritation, nerve injury and nonunion or delayed union at final follow - up . Although various reports have described freiberg's disease since 1914, classification and treatment methods thereof are not completely established.17) smillie2) classified the appearance of the metatarsal head in freiberg's disease into five stages in 1957 . In the early stages of the disease (smillie stage i to iii), fair evidence supports the use of the closing wedge osteotomy of the metatarsal head and neck.14,17) on the other hand, kinnard and lirette11,12) reported even with advanced cases there was sufficient plantar cartilage to perform the procedure . We divided our patients into early and late stages to compare the effectiveness of modified weil osteotomy among the stages.11,12,17,22) upon investigation, improvement of vas, aofas score and rom of the mtp joint did not significantly differ between early stages and late stages . Modified weil osteotomy can be a good treatment option in both early stages and late stages of freiberg's disease . Modified weil osteotomy involves open joint debridement and intra - articular dorsal closing wedge osteotomy . Open joint debridement allows removal of intraarticular pathology such as thickened synovium, loose bodies, delaminated articular cartilage and peripheral osteophytes and spurs.17) closing wedge osteotomy is a realignment osteotomy of metatarsal head and neck.18) the aim of closing wedge osteotomy is to redirect the articular surface and theoretically, dorsal closing wedge osteotomy can restore the blood supply to the metatarsal head, preventing further deformity and collaps.8,17,21) intra - articular dorsal closing wedge osteotomy enables less metatarsalgia than that of extra - articular osteotomy which often leads to excessive elevation of the metatarsal head.13) dorsal closing wedge osteotomy redirects articular surface allowing the intact plantar cartilage to articulate with the proximal phalanx . Also, dorsal closing wedge osteotomy offers similar results to extra - articular osteotomy in rom of the mtp joint . Kinnard and lirette11) reported that dorsiflexion osteotomy gave excellent results with minimal loss of mtp joint motion and with an average of 2 to 5 mm metatarsal shortening . Various fixation methods can be applied after completion of dorsiflexion osteotomy of the metatarsal, including cerclage wire,10) temporary pins,10 - 12) transosseous sutures,12) metal screw,18) dorsal t plate,9) polyglycolide pins.14) gauthier and elbaz10) suggested intra - articular dorsal wedge osteotomy fixed with cerclage wire in 1979 . The cerclage wire may cause tendinitis and temporary pins have to be removed before motion exercise . Because the remaining intact portion of the metatarsal head was too small for wire fixation, kinnard and lirette11) modified the intra - articular dorsal closing wedge osteotomy fixated with absorbable suture . Transosseous sutures can bring about loss of reduction, whereas metal screw or plate fixation after extra - articular osteotomy is more rigid . However, the metal screw or plate may be bulky and can reduce the rom of the mtp joint . Recently, absorbable polyglycolide pins have been used that do not decrease the rom of the mtp joint . After performing dorsal closing wedge osteotomy with absorbable pin fixation, lee et al.14) reported no loss and some gain in motion . Unfortunately, they are relatively weak and require multiple pinning . In this study, we fixed metatarsal head with a single screw after dorsal closing wedge osteotomy . Compared to other fixation devices such as temporary crossed kirschner wires, patients were allowed to start motion exercise and bear weight relatively early . Also, the spin screw (integra lifescience co.) with its low profile head, may reduce soft tissue irritation and has non - threaded lag for more compression . By placing the apex of the wedge as proximal as possible, a less shortened metatarsal and an enlarged distal fragment can be achieved.14) kinnard and lirette11,12) underwent an intra - articular dorsal closing wedge osteotomy in fifteen patients with predominantly late stage freiberg's disease . They reported a minimal loss of joint motion and an average metatarsal shortening of 2.5 mm . In this study, we experienced favorable improvement of rom after operation and even a floating toe occurred in 1 case . After screw fixation, foreign body reaction, fixation failure, perioperative fracture and displacement of osteotomy may occur . In our study, we could not find any complication of the sort; however, confirmation of such would require a more cases and longer follow - up . Limitations of this study include being a retrospective study, as well as no control group for being compared with dorsal closing wedge osteotomy . Some prospective and long term follow - up study with more patients and control group can prove the efficacy of dorsal closing wedge osteotomy in frieberg's disease . Through the modified weil osteotomy, we can expect offloading of the metatarsal head and restoration of mtp joint congruency . This study provides great improvement in pain and function after the procedure with a screw fixation for freiberg's disease . Modified weil osteotomy is believed to be a useful method for the treatment of freiberg's disease in both early stages and late stages.
We encountered a case where an infection with group a streptococcus (gas; ie, streptococcus pyogenes) initially caused primary peritonitis and then subsequently caused streptococcal toxic shock syndrome . The patient s life was likely saved by an emergency laparotomy followed by extensive peritoneal lavage and drainage . A 40-year - old woman was admitted to the emergency department for lower abdominal pain and numbness in the extremities . An emergency laparotomy was performed, and ascites that resembled pus and general peritonitis were noted . The patient was diagnosed with streptococcal toxic shock syndrome due to an ascending gas infection originating from vagina . Streptococcal toxic shock syndrome (stss) is a condition that suddenly and rapidly progresses as a result of an infection with group a streptococcus (gas; ie, streptococcus pyogenes). This condition can lead to septic shock, acute respiratory distress syndrome (ards), disseminated intravascular coagulation (dic), or multiple organ failure, which accounts for its high fatality rate . As reported, we encountered a case where gas initially caused primary peritonitis and subsequently caused stss . In the case in question, the patient s life was likely saved by performing an emergency laparotomy followed by extensive peritoneal lavage and drainage . A 40-year - old woman was admitted to the emergency department of aomori prefectural central hospital for lower abdominal pain and numbness in the extremities that developed 14 hours prior . She was on day 6 of her menstrual cycle, had a temperature of 37.9 c, a blood pressure of 92/46 mmhg, a heart rate of 97 beats / min, and an oxygen saturation of peripheral artery (spo2) of 96% . Blood test results were as follows: white blood cell (wbc) count of 8100/l, hemoglobin (hb) of 6.2 g / dl, platelet (plt) count of 46.2 10/l, c - reactive protein (crp) of 9.61 mg / dl, blood urea nitrogen (bun) of 20.1 mg / dl, and creatinine of 1.52 mg / dl . Uterine fibroids, ascites, and thickening of the peritoneum were suggested, and intraabdominal hemorrhage was also a possibility, but computed tomography (ct) scan failed to reveal any obvious sources of bleeding . A pelvic infection was suspected, so the patient was admitted and azithromycin (azm) 500 mg / d was started . However, 29 hours after initial presentation, abdominal pain intensified and the patient s temperature increased to 38.4 c . The patient continued to exhibit hypotension, breathing difficulties, and spo2 of 90% or lower . An intraabdominal hemorrhage or formation of a pelvic abscess was suspected after 32 hours of initial presentation, and the decision was made to perform an emergency laparotomy . Blood test results just prior to surgery were wbc count of 700/l, hb of 7.8 g / dl, plt count of 10.2 104/l, crp of 25.5 mg / dl, bun of 35.2 mg / dl, and creatinine of 2.12 mg / dl . A large volume of ascites that resembled pus and general peritonitis were manifested in the peritoneal cavity . Other than uterine fibroids, lesions were not evident in the uterus, ovaries, or fallopian tubes . The intestines were somewhat edematous, but no gastrointestinal problems, such as a gastrointestinal perforation and cholangitis, were noted . 1). Peritoneal lavage and drainage was performed along with a partial omentectomy and an appendectomy . A peritoneal drain was placed and the abdomen was closed . While she was managed on a ventilator, 1 g of meropenem three times a day (tid) was added to azm . The total volume of transfusions from surgery until discharge from the intensive care unit was 24 units of packed red blood cells, 22 units of fresh frozen plasma, and 60 units of platelets, continuous hemodiafiltration was performed, direct hemoperfusion was performed with a polymyxin b - immobilized column, an immunoglobulin preparation was administered, and hemodynamics was managed with continuous intravenous infusion of noradrenaline . Blood test results about 10 hours after surgery revealed the following: wbc count of 21,000/l, hb of 10.1 g / dl, plt count of 4.6 104/l, crp of 21.5 mg / dl, bun of 36.4 mg / dl, creatinine of 2.15 mg / dl, total bilirubin of 1.83 mg / dl, aspartate aminotransferase of 699 iu / l, alanine aminotransferase of 226 iu / l, fibrin degradation product of 93.3 g / ml, fibrinogen of 204 mg / dl, and prothrombin time of 24.6 seconds . Chest x - rays revealed infiltrated in the fields of both lungs, and ards, dic, and multiple organ failure were manifested . On day 4 after initial presentation, gas was isolated in an intraoperative peritoneal fluid culture, resulting in the diagnosis of stss . The bacteria isolated in blood cultures, vaginal culture, or urine culture after administration of azm were not the same . In addition, 900 mg of clindamycin tid and four million units of penicillin g once a day (sid) were administered . On day 4 after initial presentation, a rash resembling erythema developed primarily in both lower extremities and necrosis developed in the distal extremities due to poor peripheral circulation . Histopathology indicated necrotic tissue, and gram staining revealed gram - positive bacteria (fig . Stss is a reemerging infectious disease that was initially reported in the us in 1987.1 stss has a fatality rate of 40% or higher.1 gas infection has long been known to cause pharyngitis, tonsillitis, scarlet fever, and sequelae, such as rheumatic fever and acute glomerulonephritis . Initial manifestations of stss include pain in the extremities, swelling, a fever, and hypotension; soon after developing stss (within 2448 hours), it can cause acute renal failure, ards, dic, and soft tissue necrosis, and it can even lead to death.2 the virulence factors of gas are far more varied than those of other bacteria, and the various exotoxins produced by gas stimulate cytokine release and act as superantigens.3 a study of stss isolates has suggested that specific genetic mutations allow gas to evade elimination by the host s neutrophils, causing a highly pathogenic disease characterized by fulminant infection.4 according to a systematic review of 32 cases of primary peritonitis due to stss, the median age for the development of stss was 38 years (range: 2387 years), female patients outnumber male patients at a ratio of 4:1, and 91% of patients were healthy with no underlying illness.5 according to the same systematic review, 16% of patients developed stss from an ascending infection originating from vagina, 6% of patients developed it from pharyngitis, 9% of patients developed it from a droplet infection, and 69% of patients developed it via an unknown route.5 in the current case, the patient was transported to the emergency department 14 hours after developing symptoms . When the patient was seen, she had a slight fever, and her vital signs stabilized, so stss would have been an unlikely differential diagnosis . There is a room to discuss the appropriateness of performing an emergency laparotomy, while the patient had a wbc count as low as 700/l . Peritoneal fluid was tested for bacteria about 70 hours after initial presentation, and the results revealed the presence of gas . This approach saved the patient s life, so performing surgery was the correct choice in this case . Routes of infection have often cited an ascending infection originating from vagina.5 in the current case, infection occurred during menses, and a pedunculated tumor stained positive for gram - positive bacteria, so the patient presumably had an ascending infection originating from vagina . If a patient complaining of lower abdominal pain has a fever or elevated inflammatory markers, further testing (which includes vaginal culture) must be performed prior to the administration of an antibiotic . A recent report demonstrated a 6-year - old girl who initially presented with presumed viral gastrointestinal infection for a week and later developed catastrophic primary peritonitis and septic shock requiring resuscitation and emergency exploratory laparotomy due to group a streptococci.6 the incidence of stss has increased over the past few years, so stss must be kept in mind as potential diagnosis if a patient has abdominal symptoms that abruptly worsen.
Around the time of independence, the metaphor of madness is best exemplified by pandit jawaharlal nehru, who, in his address to the nation on january 31, 1948, the morning after the assassination of mahatma gandhi, said, a madman has put an end to his life, for i can only call him mad who did it . Events between 1857 and 1947 brought to the fore, both metaphorical concepts of madness and sanity as well as psychopathological manifestations of contemporary political preoccupations . In 1857, john nicholson planned his long march from his cantonment in punjab to delhi, to recapture delhi and overthrow the last mughal emperor, bahadur shah zaffar, who had become the center for the first war of independence . Nicholson was an irishman, an army commander, rather colorful, someone who could be called almost psychopathic . He had allegedly impressed the sikhs so much with his reckless valor and dash, that he had a sect named after him as nickel - seyni's, who were prepared to obey his orders to the end . A year earlier, while at bannu in the north - west frontier, when a man with a long sword had rushed him one morning, nicholson, grabbing a musket from one of his guards, ordered him to put the sword down . When the man refused, and insisted that either he or i must die, nicholson shot him dead, and commented in a letter that the poor wretch turns out to be marwutee who had been religiously mad for some time his spiritual instructor disappeared entirely, and i am afraid, gone into the hills . In the aftermath of 1857, in addition to the various hangings, beheadings, and blowing away from cannons, some individuals did find their way into lunatic asylums . As a soldier visiting the asylum in benares on august 24, 1861 described: among the many idiots there confined was one calling himself a pundit, who talked so fast and long as to the destruction of the europeans in india that one would almost credit him with sanity, were he not known to be otherwise; vehement in his manner of delivery as to his determination in regard to all white faces he certainly must have had some knowledge of what had transpired in the mutiny and as we saw that he desired to continue his conversation on that topic the warder cut him short by ordering him into his cell and locking him up . H, an irishman living in delhi, began saying that he had become a duke, and that his pay had been stolen by the british government and spent on buying oranges (!). He converted to islam at the jama masjid, and took the name ahmed din . He said he was on the way to kabul, as the russians were expected to help the muslims to overthrow the british . He exhorted the muslims to unite, and get ready for the event, and meet the russians in peshawar . Across the road, in the garrison at the red fort, the british became very worried . H had a deeper knowledge of the great game of kipling, or a prescient vision of the afghanistan and south asian power games of the europeans is unclear! This caused much consternation, and furore in delhi, because it was widely believed that he had been labeled insane for deciding to convert from christianity to islam, rather than the other way around . The population of delhi was thought to be highly excitable, as the events of 1857 had shown, and it was thought prudent to not keep him in the delhi asylum . He was shifted to the asylum at colaba, bombay (where there was a small asylum for the british, en route to the asylums in the uk) maintained by the east india company . The authorities perhaps realized the political impact of certain ideas, even though the source of these ideas could be defined to be bereft of reason . In contrast, another patient in the delhi asylum wanted to help queen victoria, by interceding in the north west, and rule kabul on her behalf . Mr . Imrit, in the delhi asylum in 1883, claimed he was the descendant of the sen dynasty (a hindu dynasty that ruled bengal in the 1112 centuries). He said that he had leased india on contract to the british for 80 years, after which he would divide it between the russians and the chinese (predating the dissensions among bengals intellectuals between marxist and maoist ideas by a century). Habib shah, who was discovered one morning in 1883 on the emperors throne at the diwan - e - am in the red fort, claiming to be the emperor, and the last of the mughal dynasty as he had been sent by nizam - ud - din (the sufi patron saint of the mughal empire). How he reached there, the fort being an army encampment, remained a mystery . In any case, when rulers call each other mad, madmen have every right to be rulers too!! Whether the great game had already seeped into the consciousness of the community, including those who were admitted into the asylums; or was it the creation of madmen that spread from the asylums into the community is still open to conjecture . Ideas about the independence of india gradually spread all over india and even in in the kingdom of mysore, patients were to occasionally describe such preoccupations . As the politics of the region became more fractious and unreasonable, the growing communal problem was often viewed as a sign of madness . A tract written in 1943 set about describing the various machinations going on as the political insanity of india (vakeel 1943). Commenting on the orgy of vituperation, slogans, alternating threats of civil disobedience and retirements into wilderness indulging in torrents of verbosity vakeel bemoans that by exploiting passions on all sides a few are guiding the many to disaster as they are responsible with their fantastic hallucinations and interpretations of freedom and slavery for a good deal of unhappiness in the country . He was very worried that in the near future, many would suffer for the sins of a few and warned that what i am writing is portentous a few years later, when all that had been predicted had indeed come to pass, many commentators did indeed invoke the vision of madness as being responsible for the violence during the partition . Early in september 1947, gandhi referred to the violence following the declaration of independence as let it be said of them that the inhabitants of delhi had gone mad temporarily but that sanity had now returned . Since this recovery was short lived and frequent relapses of pointless violence and intemperate speech occurred, he again issued a plea for a return to sanity, a few days before he was assassinated . A reporter covering the funeral of mahatma gandhi, and sitting with nehru on the deck of the gun carriage that formed the cortege, commented on how the procession was delayed on the banks of the yamuna, by a madman dancing in front of the carriage . The whole procession came to a halt, while the man was (presumably kindly) led, away . Many ideas that have guided recent history were once thought to be inimical to social order, even dangerous, or symptoms of madness . We are all familiar with the persecution of galileo for overturning the shared belief that the sun revolved around the earth, and the many ideas of newton which transformed the world . Newton formally believed in alchemy, and wrote principia mathematica during a prolonged period of seclusion, allegedly an episode of nervous breakdown . The world of politics is also well populated by accounts of the madness of kings, but it is the madness of the people often reflects the madness of their kings . Such notions of what stands in the realm of reality, wishful thinking, imagination and what borders on over - valued ideas or even delusions are resonant in contemporary times . Several books with titles such as imagining india and re - imagining india have emerged over the past two decades . The borders between imagination and delusion being somewhat hazy, as these accounts show, in the clinical context, they could obviously be of concern, particularly in political and religious contexts . As pope pointed out in the 19 century, that being irrational, the insane though in the state are not of the state . On the other hand, although not of the state the insane are yet in the state.9 in view of this complexity, it is only logical that the borders between madman and the state are somewhat permeable, and both their histories intertwined . The work was supported by a grant from the wellcome trust turning the pages (wt096493ma). The work was supported by a grant from the wellcome trust turning the pages (wt096493ma).
Adenoidal hypertrophy is a common condition in children and can cause symptoms such as mouth breathing, nasal discharge, snoring, sleep apnea, and hyponasal speech it also contributes to the pathogenesis of rhinosinusitis, recurrent otitis media, and otitis media with effusion . Adenoid curette guided by an indirect transoral mirror and a headlight is a simple and quick procedure that has already been in use for a long time, but this method carries a high risk of recurrence unless done by a well - experienced surgeon [3, 4]. Recent methods, such as curved suction electrical coagulator and the curved microdebrider shaver transorally [4, 5] guided by a transoral indirect mirror or a 45-degree endoscope, have successfully been used . Becker et al . Removed the adenoidal tissues transnasally combined with transorally under endoscopic visualization . Koltai et al . Described a power - assisted adenoidectomy technique without the use of nasal endoscope while yanagisawa and weaver used the same technique under endoscopic vision but they found difficulty in maneuvering the microdebrider tip into the nasopharynx . Each of these methods has its advantages and disadvantages; however, the symptoms of adenoid hypertrophy may recur or even persist after removal of the adenoid . In this paper, we described an easy transoral endoscopic removal of adenoidal tissues aiming to assess the efficacy of this method in relieving the obstructive nasal symptoms . Three - hundred patients were included in this study, all had adenoid (with or without tonsillar) hypertrophy . One hundred eighty seven were males and one hundred thirteen were females, their ages ranged between three to seven years with a mean age of four years and four months . All cases were collected from the otolaryngology clinics of kasr el - aini and abu el - rich children hospital of cairo university in the period from january 2003 to december 2006 . Informed consents were obtained from the parents of the patients, and the principles outlined in the declaration of helsinki were followed . Cases missed followup were excluded . Under general anesthesia with oral endotracheal intubation, a boyle - davis mouth gag was used to open the mouth . The soft palate was retracted with bilateral rubber catheters passed from the nose to the mouth and the two ends clamped tightly by artery forceps . The 70 hopkins 4-mm nasal endoscope is introduced through the mouth, and the adenoid mass is identified (figure 1). A camera mounted on the endoscope and the endoscopic view curettage of the main adenoid mass was carried out using adenoid curette, with removal of residual adenoidal tissues using st claire thomson forceps while suction is used to clear the field, the bleeding is usually minimal especially after complete removal of all nasopharyngeal lymphoid tissues . At the end of the procedure, a pack of gauze was inserted into the nasopharynx for few minute or till removal of the tonsils (if indicated). Tonsillectomy was carried out for seventy children and myringotomy with insertion of ventilation tubes was carried out for one - hundred and four cases . Routine postoperative treatment in the form of antibiotics and analgesics for one week was given . One year after the operation, the parents or caregivers of all children were contacted by telephone . They were asked to answer if the preoperative obstructive symptoms recurred or not . Those children with recurrent symptoms at the time of interview were invited to undergo flexible nasopharyngoscopy, and if they were found to have adenoid regrowth, we planned to search for the cause . The study sample (n = 300) had a mean age of 4 years and 4 months at the time of the adenoidectomy . No cases presented with postoperative complications such as hemorrhage, airway problems, grisel's syndrome, stenosis, or velopharyngeal insufficiency . Telephone questionnaire showed recurrent obstructive symptoms in one case only; the child and his parents were invited for assessment . He was a male child aged 5 years and 3 months and he underwent adenoidectomy without tonsillectomy accompanied with myringotomy and ventilation tube insertion . His parents stated that the child started to snore 5 months after removal of his adenoid; the father gave a history of allergic rhinitis and bronchial asthma . Flexible nasopharyngoscopy for this patient showed recurrent obstructive adenoid, mainly in the right side of the nasopharynx . Because of family history of allergic rhinitis, the child was subjected to skin prick test and he showed positive result with dust mite allergens (dermatophagoides pteronyssinus and dermatophagoides farinae; tests 708 and 725 manufactured by allergopharma co.). The child was subjected to revision transoral endoscopic adenoidectomy with antiallergic treatment; he showed no recurrent obstructive symptoms till the end of the study . Removal of adenoid using transoral mirror is the most commonly used method; although the indirect visualization of adenoidal tissue, the incidence of recurrence is not known . In this study, we described an easy transoral endoscopic removal of adenoidal tissues using the adenoid curette and st claire thomson forceps . The advantage of this method is the direct visualization of the operative field which would decrease the incidence of residual lymphoid tissues, also it helps to avoid eustachian tube injury that may causes its fibrosis . Moreover, this procedure dose not need expensive equipments as all used instruments are already present in the operating theatre, it may takes 2 - 3 minutes more than the conventional mirror method . However, transoral endoscopic removal may need some experience for junior surgeons that are usually unacquainted with the transoral view, moreover the wear and tear of the endoscope with each use may increase the expenses . Schaffer and wong stated that there is a short learning curve when applying endoscopic sinus techniques to the adenoidectomy procedure, but they believe otolaryngologists will find transoral endoscopic adenoidectomy to be easily learned and clinically successful, with little chance of adenoid regrowth . Wan et al . Reported that teaching the transoral endoscopic adenoid removal to trainee surgeons is much easier when combined with the video monitoring facilities . Recurrent adenoids are most probably due to regrowth of residual lymphoid tissues left as a result of blind removal . However, buchinsky et al . Commented that adenoids rarely, if ever, regrow enough to cause symptoms of nasal obstruction after adenoidectomy that includes visualization and electrocautery of the adenoid bed . Our study showed recurrent adenoid in 1 out of 300 cases (0.33%); schaffer and wong used a similar technique for adenoid removal and they reported recurrence only in 2 patients out of 4000 adenoidectomies performed . However, many authors [12, 14] reported that recurrent adenoid after its removal may be attributed to nasal pathology, so we suggest that nasal allergy may play a role in recurrence in our case . Used the 4-mm 0 telescope transnasally at the end of the routine transoral adenoidectomy and they found residual adenoid tissue in all cases of their 236 except for 12 cases (5.1%); therefore they stressed that direct visualization is very important during the procedure . Wan et al . Performed transoral curettage adenoidectomy guided with trans - nasal endoscope on 13 children, they reported marked improvement with no recurrence of obstructive symptoms . However, the sample of their patients was small and the procedure is very difficult as introduction of the curette into the nasopharynx may be accompanied with bleeding that obscures the view, while our method is much easier as the palate was retracted by 2 catheters to open the nasopharynx facilitating insertion of the curette above the superior border of the adenoid . In the technique of we cannot retract the palate while the nasal endoscope is passing through the nose as the child's nose is very difficult to accommodate a catheter and an endoscope . Kamel and ishak described trans - nasal endoscopic removal of the adenoid using the instruments of endoscopic sinus surgery, and they achieved improvement in 94% of their cases . Partial transnasal endoscopic adenoidectomy has been described in cases that complained of obstructive adenoid with submucous cleft palate [17, 18]. The advantage of the endoscopic vision in these cases is to avoid complete adenoid removal that may results in velopharyngeal insufficiency . None of our cases developed postoperative complications, as direct well vision would decrease haemorrage and if it happened it can be easily controlled . It also decreases the chance of injury of eustachian tube orifices and violent removal of normal nasopharyngeal tissues that may result in fibrosis and stenosis . This study was designed to evaluate the efficacy of transoral endoscopic removal of the adenoid, and it does not address the superiority of this method over other methods . To address this issue however, we strongly advise randomized trials to compare this method with other methods regarding the operative time, the amount of intraoperative blood loss, the postoperative complications, and the incidence of recurrent symptoms . In conclusion, transoral endoscopic adenoidectomy is the recent advancement of classic curettage adenoidectomy with good direct vision of the nasopharynx that enables the surgeon to avoid injury of important structures as eustachian tube orifices and also gives him the chance to completely remove the adenoidal tissues.
B lymphocytes produce immunoglobulins consisting of a heavy chain and either a kappa (igkc) or a lambda (iglc) light chain . Each b lymphocyte decides early by the rearrangement of its immunoglobulin genes which light chain to produce . The excluded light chain gene is not properly rearranged or remains in germ line configuration . In healthy humans 6070% of the b cells produce kappa chains and the rest produce lambda chains [1, 3]. Normal lymphoid tissues therefore contain a mixture of b cells that express igkc and iglc at a ratio of about 60: 40 = 1.5 . Tumors of b cell origin are monoclonal and arise from one transformed cell . The single cell origin of malignant clones results in exclusive expression of igkc or iglc light chains in the vast majority of all b cell malignancies although b cell tumors that produce both kappa and lambda chains have been reported . The clonal expression of igkc or iglc is thus used as an important diagnostic marker for b cell malignancies and currently determined on protein level by immunohistochemistry (ihc), flow cytometry (fc), or enzyme - linked immunosorbent assay techniques . Previously, we used reverse transcription quantitative real - time pcr (rt - qpcr) to quantify igkc and iglc gene transcripts in a small set of lymphomas and found that also gene expression level clonality was frequently evident . In the present study we have used the same rt - qpcr method together with ihc and fc to analyze a larger cohort of 39 non - hodgkin lymphomas, 16 chronic lymphatic leukemias, and 5 b cell derived tumor cell lines . The non - hodgkin lymphomas consisted of 20 diffuse large b cell lymphomas, 16 follicular lymphomas, and 3 mantle cell lymphomas . The samples were transported from the operation theatre in ice - water - chilled boxes, handled in the laboratory within 30 min, and stored at 140c . Parts of the tissues were fixed in formalin and embedded in paraffin according to the routine protocols of the pathology laboratory . Diagnosis was reached by a combination of microscopic histological evaluation, ihc of several markers, including the and chains, and in some cases by fc . Series of 5 m tissue sections were cut from each biopsy, deparaffinized, rehydrated, and stained with the following antibodies: rabbit anti - human kappa light chains and rabbit anti - human lambda chains (a0191 and a0194, dako). The antibodies were used at a dilution of 1: 400 and bound antibodies were visualized using the second labeled antibody streptavidin biotin peroxidase system (dako). Stained sections were examined with a light microscope . For fc, a routine protocol for preparation of lymphoid cells from lymphoma tissue was used . Direct labeled antibodies specific for immunoglobulin kappa and lambda chains (a0191 and a0194, dako) were used for staining of recovered cells . First a gate for lymphocytes was set using forward and side scatter, followed by gates for kappa and lambda staining cells . We used a kappa: lambda ratio less than 0.4 or greater than 5 to prove fc monoclonality . The samples were classified according to the revised european - american lymphoma and world health organization classification system . Rna was extracted by use of the fast prep system (fastrna green; qbiogene). We mixed 10 g of total rna with 2 g of poly (dt) oligomers (pharmacia) and incubated the mixture at 65c for 5 min . First - strand cdna synthesis was performed by adding 0.05 mol / l tris - hcl (ph 8.3), 0.075 mol / l kcl, 3 mmol / l mgcl2, 0.01 mol / l dithiothreitol, 10 u / ml moloney murine leukemia virus reverse transcriptase (life technologies), 0.05 units / ml rna guard (life technologies), and 10 mmol / l of each deoxyribonucleotide (life technologies) to a final volume of 20 l and incubating the samples at 37c for 1 h. the reaction was terminated by incubation at 65c for 5 min, and samples were stored at 80c . Real - time pcr measurements were performed on a rotorgene 3000 (corbett research). Each pcr reaction contained 10 mmol / l tris - hcl ph 8.3, 50 mmol / l kcl, 4 mm mgcl2, 400 mol / l each dntp, 300 nmol / l each primer, 0.04 u/l jumpstart taq (sigma), and 0.25x sybr green (molecular probes) in a 20 l reaction volume . The temperature profile was 95c for 3 min followed by 40 cycles of amplification (95c for 20 sec, 60c for 20 sec, and 72c for 20 sec). Formation of correctly sized pcr products was confirmed by agarose gel electrophoresis for all assays and melting curve analysis for all samples . A 95% confidence region for the igkc: iglc ratio using the negative lymphadenitis was used to prove rt - qpcr monoclonality . Fragments were excised from the gels and used as templates for dna sequencing as previously described . The samples were analyzed by ihc, fc, and rt - qpcr for expression of igkc and iglc light chains (tables 1 and 2). Ihc indicated an exclusive or heavily dominant expression of igkc or iglc chains indicative of a monoclonal origin in all but one analyzed samples (98%). Rt - qpcr analysis indicated a monoclonal dominance in 33 of 55 (60%) analyzed samples, while fc scored monoclonal dominance in 30 of 39 (77%) analyzed samples . Thus the ihc method was superior to fc and rt - qpcr in detecting monoclonality . The divergent results may in part be explained by the fact that biopsies, besides the tumor cells, often contain considerable numbers of normal lymphocytes that could contribute to the rna samples analyzed by rt - qpcr and the cell population studied by flow cytometry . Ihc analysis, on the other hand, allows for selective analysis of representative parts of the tumor tissue thus avoiding contribution of normal b lymphocytes . Furthermore, it is also known that 6070% of non - hodgkin lymphomas lack surface expression of iglk and iglc . However, there was an overall good correlation in expression levels of igkc and iglc between fc and rt - qpcr data despite the fact that fc data reflects cell number and rt - qpcr data reflects transcript numbers (figure 1). In two cases (samples 135 and 168, table 1), rt - qpcr detected clonal populations where fc failed . Rt - qpcr may therefore serve as a valuable complement to ihc and fc in detection of monoclonal b cell populations . Moreover, rt - qpcr analysis can also be employed on minute fine needle aspirate samples that are not sufficient for flow cytometry or ihc and the same sample may also be analyzed simultaneously for expression of more marker genes . Several of the igkc and iglc cotranscribing tumors appeared by ihc and fc to consist of homogenous tumor cell populations with no or few normal lymphocytes suggesting that some malignant b cell clones were dual producers of light chain mrna (figure 2). Dual production of iglc chain mrnas and proteins has been reported for various types of malignant b cell clones and normal b lymphocytes [4, 9]. These reports and our data prompted us to analyze five monoclonal b cell derived tumor cell lines (table 3). These cell lines produce only one light chain species as analyzed by immunofluorescence, but four of them contained both igkc and iglc transcripts . To further confirm this result we isolated pcr products from two of the cell lines and sequence analysis showed that igkc and iglc transcripts were indeed produced simultaneously in these cell lines . Thus we conclude that human monoclonal b - lymphoid populations frequently simultaneously produce igkc and iglc transcripts . Dual producers were most common in the follicular lymphoma group in which 11/16 tumors failed to score for monoclonality based on rt - qpcr analysis . This could possibly be explained by larger abundance of normal cells in follicular lymphomas since fc showed highest failure rate for these samples, and may also depend on a higher frequency of dual producers . Eleven of twelve cases where monoclonality was not detected by rt - qpcr produced igkc protein as judged by ihc . This is more than expected from the normal ratio of igkc and iglc producers among normal b lymphocytes and lymphoma populations . In the present cohort of thus, it is possible that dual mrna producers arise more often among kappa protein producing cells . This is surprising since kappa chains are considered to be rearranged first and iglc rearrangement is pursued only if igkc rearrangement fails to produce a functional igkc protein . The activation of iglc transcription is most likely a result of rearrangement that occurs after igkc protein is produced and may therefore indicate a failure to inactivate the rearrangement mechanism . Failure to regulate a potentially risky dna rearrangement process may also contribute to the frequent multiple genetic rearrangements in lymphomas . Age and gender were not correlated to the determined clonality (data not shown). We conclude that ihc, fc, and rt - qpcr analysis of human b - lymphoid tumors gives partially divergent results . This may in part be explained by contribution of normal b lymphocytes into the tumor tissue . Our results also suggest that dual transcription of igkc and iglc loci is common in these tumors . This has to be taken in consideration when rt - qpcr analysis of igkc and iglc is used for diagnostic purpose . Rt - qpcr remains an interesting complement for diagnostic purpose when only small samples, such as from fine needle cytology, are available.
Angiomyxomas are benign, locally infiltrative mesenchymal neoplasms with a predilection for the female pelvis and perineum in their reproductive age group . These tumours often reach too large dimensions before becoming clinically symptomatic and they usually tend to recur after excision . Accurate preoperative diagnosis should alert the surgeon to the need for wide excision, which is essential for prevention of local recurrence . This is easily done with robotic assistance due to its advantages like three dimentional vision and advanced degree of freedom within the pelvic cavity . We report here a similar case presenting as a retrovesical tumor in a male patient, which was excised with robotic assistance . A 62-years - old male presented to us with mild obstructive lower urinary tract symptoms (luts) since last 1 year, with a history of acute retention of urine 3 month back with failed catheter free trial . He underwent holmium laser enucleation of prostate (holep) elsewhere with histopathology reporting as benign prostatic hyperplasia . Patient failed to void after catheter removal and on further evaluation he was found to have pelvic mass . Digital rectal examination revealed a cystic mass simulating benign prostatic hyperplasia (grade 3). Magnetic resonance imaging (mri) pelvis revealed a well - marginated lesion within the prostate gland resulting into significant prostatomegaly with no extra prostatic extension [figure 1]. Transrectal biopsy was done, which was inconclusive with no identifiable prostatic tissue . In view of discrepancy between mri report and biopsy report we performed cystoscopy which showed a hump in the prostatic urethra extending into the trigone, with prostatic lobes not visible (because of previous holep). Tumor was ~6 cm 4 cm 2.5 cm in size [figure 2]. We were not able to separate the mass from bladder due to difficulty in entering into proper plane . Hence we opened bladder deliberately and resected from within the bladder, closed cystostomy and drained bladder . Histo - pathological examination [figure 3] revealed moderately cellular tumor composed of sheets of spindle to stellate shaped cells with vascular channels in between . On immunohistochemistry cells were positive for desmin, focal positivity with smooth muscle antibody (sma), s100 and ki-67 . Magnetic resonance imaging showing a well - marginated lesion within the prostate gland resulting into significant prostatomegaly, with no extra prostatic extension . Also gross specimen of the tumor with gelatinous appearance and size of ~6 cm 4 cm 2.5 cm . Histopathology revealed moderately cellular tumor composed of sheets of spindle to stellate shaped cells with vascular channels in between . On immunohistochemistry cells were positive for desmin, focal positivity with smooth muscle antibody, s100 and ki-67 . Aggressive angiomyxoma was first described in 1983 by steeper and rosai, and fewer than 150 cases have been reported in the world medical literature . The recurrence rate is high, and often extensive resections are performed with considerable morbidity . In men, the tumour involves analogous sites including the scrotum and inguinal area and usually appears at an older age . There might be a relation with hormonal status that might explain a female to male ratio of slightly more than 6:1 . These lesions are characterized as soft, non - encapsulated tumours with finger - like projections infiltrating the surrounding soft - tissues . The tumour grows slowly and is benign as suggested by the histology and by the fact that it shows no tendency to metastasize . The low attenuation at unenhanced computed tomography (ct) and high signal intensity at t2-weighted mri are consistent with a myxoid matrix, high water content within the mass, or both . The enhancement of t1-weighted mri after administration of gadolinium is related to the vascularity of aggressive angiomyxoma . There is no evidence of gross fat within the mass, which distinguishes this tumor from other fat - containing tumors that also occur in the pelvis of women . Surgery is usually the first line of treatment (open, laparoscopic or robotic assisted), radical surgery with wide margins and long - term follow - up is advised . Hormonal suppression seems to be a plausible treatment option because these tumours occur predominantly in premenopausal women of reproductive age, may grow rapidly during pregnancy and have been shown to express immunohistochemical positivity for oestrogen and progesterone receptors . Their surgery is challenging because of the infiltration and the difficult dissection . In the past, most authors advocated wide excision along with genito - urinary and digestive tract resections if necessary . Most recently robotic assisted laparoscopic excision has been promising, as seen in our case . Male angiomyxoma, athough a rare benign tumour, should be considered in the diferential diagnosis of pelvic mass in a male patient presenting with severe obstructive luts, and robotic assisted excision should be utilised wherever available.
Although recent advances in the medical and surgical treatment of spinal lesions have led to better outcomes, adhesive arachnoiditis is still one of the most serious conditions . Adhesive arachnoiditis may result in the formation of syringomyelia or myelomalacia, causing neurological deterioration such as sensory disturbances in the extremities, urinary disturbance, or sexual dysfunction, all of which affect activities of daily living (adl). Various surgical procedures, including cerebrospinal fluid (csf) shunting or subarachnoid reconstruction, are available. [14] shunting of csf from the syringomyelia to the subarachnoid, pleural, or peritoneal space has been proposed to resolve the propagation of the syringomyelia or myelomalacia, but the effect of the procedure is not long lasting and revision surgery is needed rather frequently. [46] surgical outcome may be limited by the high rates of neurological recurrence . Microsurgical dissection of the arachnoid adhesion, with decompression of the subarachnoid space, is another approach and may achieve better and more long - lasting outcomes in selected cases . A clear indication as well as surgical procedures that take into account the underlying pathophysiology are absolutely necessary . Here, we emphasize the importance of imaging diagnosis of focal adhesive arachnoiditis of the spinal cord and describe the surgical technique of microsurgical arachnoidolysis to mobilize the spinal cord and reconstruct the csf pathway . Between january 2008 and the present, four consecutive patients with symptomatic syringomyelia or myelomalacia caused by focal adhesive arachnoiditis underwent surgical treatment at the osaka city university hospital [table 1]. The four patients satisfied our inclusion criteria, i.e., (1) medical history of spinal trauma, surgery, or infection; (2) clear symptoms and objective neurological deficit; and (3) clear mri visualization of syringomyelia or myelomalacia with the suggestion of focal adhesive arachnoiditis . Clinical summay of the patients with focal adhesive arachnoiditis of spinal cord assessment of the the clinical condition before and after surgery was based on the modified mccormick adl grading system and the sensory scoring system[table 2]. An mri study was conducted in each patient before surgery in order to carefully analyze the location and distribution of the adhesive arachnoiditis . The focal adhesive lesion was assessed using constructive interference in steady - state (ciss) mri or myelographic mr imaging using true fast imaging with steady - state precession (truefisp) sequences . Based on the preoperative assessment, surgery was indicated in these patients with syringomyelia or myelomalacia caused by focal adhesive arachnoiditis . Patients were examined 3 months after surgery for clinical and radiological assessment, of improvement and were then followed up at regular intervals . Modified mccormick activities of daily living grading system and sensory scoring system the patient was placed in the prone position under general anesthesia . The patient's thorax was elevated 15 and the position of the head was maintained using three - point head fixation to prevent inadvertent cervical flexion and pressure on the face . All pressure points were securely padded to avoid any venous congestion or peripheral nerve injury . Somatosensory - evoked potential (sep) or motor - evoked potential (mep) monitoring was applied in case the patient developed significant myelopathy . Trains of transcranial electrical stimulation over the motor cortex were used to elicit meps from limb target muscles . The operation was paused in every case when there was significant sep or mep changes . Surgical exploration took place at a minimum of four or five spinal levels, with the arachnoid adhesive lesion in the center of the surgical field . The dura mater was incised carefully so as not to injure the arachnoid membrane until normal arachnoid tissue and spinal cord appeared [figure 1a]. The arachnoid membrane was first opened rostral and caudal to the lesion to secure the normal subarachnoid layer . The arachnoid dissection was started on the dorsolateral side of the spinal cord with resection of the dentate ligament at all exposed levels [figures 1b c]. The thickened arachnoid tissue around the nerve roots in particular was dissected as far as possible to mobilize the spinal cord . The thickened arachnoid tissue was removed or resected, not in an en bloc fashion but fragment by fragment, so that some fragments of the thickened arachnoid tissue were left behind on the dorsal surface of the spinal cord . Next, a ventral dissection was started from both sides, with avoidance of excessive manipulation of the spinal cord [figures 1d e]. The ventral dissection was finally accomplished and pulsatile movement of the spinal cord could be visualized [figure 1f]. In all cases, a small rubber sheet was introduced on one side, passed below the spinal cord, and removed from the other side to ensure that the spinal cord was completely mobilized . After the dura mater and spinal cord were free from adhesions, an expansive dural plasty was performed using autologous fascia lata [figure 2a], followed by an expansive laminoplasty of the lift - up style [figure 2b]. Intraoperative photographs showing the surgical steps of the microsurgical arachnoidolysis in the procedure in case 1 . (b) higher magnifi cation of the boxed area in a revealed the thickened arachnoid tissue on the dorsolateral side of the spinal cord . (d) and (e) a ventral dissection was started from both sides with avoidance of excessive manipulation of the spinal cord . (f) the pulsative movement of the spinal cord was visualized at the fi nal step . Intraoperative photographs showing the dural plasty using autologous fascia lata (a), which was followed by expansive laminoplasty of lift - up style using hydroxyapatite laminar spacers (b). Between january 2008 and the present, four consecutive patients with symptomatic syringomyelia or myelomalacia caused by focal adhesive arachnoiditis underwent surgical treatment at the osaka city university hospital [table 1]. The four patients satisfied our inclusion criteria, i.e., (1) medical history of spinal trauma, surgery, or infection; (2) clear symptoms and objective neurological deficit; and (3) clear mri visualization of syringomyelia or myelomalacia with the suggestion of focal adhesive arachnoiditis . Assessment of the the clinical condition before and after surgery was based on the modified mccormick adl grading system and the sensory scoring system[table 2]. An mri study was conducted in each patient before surgery in order to carefully analyze the location and distribution of the adhesive arachnoiditis . The focal adhesive lesion was assessed using constructive interference in steady - state (ciss) mri or myelographic mr imaging using true fast imaging with steady - state precession (truefisp) sequences . Based on the preoperative assessment, surgery was indicated in these patients with syringomyelia or myelomalacia caused by focal adhesive arachnoiditis . Patients were examined 3 months after surgery for clinical and radiological assessment, of improvement and were then followed up at regular intervals . Modified mccormick activities of daily living grading system and sensory scoring system the patient's thorax was elevated 15 and the position of the head was maintained using three - point head fixation to prevent inadvertent cervical flexion and pressure on the face . All pressure points were securely padded to avoid any venous congestion or peripheral nerve injury . Somatosensory - evoked potential (sep) or motor - evoked potential (mep) monitoring was applied in case the patient developed significant myelopathy . Trains of transcranial electrical stimulation over the motor cortex were used to elicit meps from limb target muscles . The operation was paused in every case when there was significant sep or mep changes . Surgical exploration took place at a minimum of four or five spinal levels, with the arachnoid adhesive lesion in the center of the surgical field . The dura mater was incised carefully so as not to injure the arachnoid membrane until normal arachnoid tissue and spinal cord appeared [figure 1a]. The arachnoid membrane was first opened rostral and caudal to the lesion to secure the normal subarachnoid layer . The arachnoid dissection was started on the dorsolateral side of the spinal cord with resection of the dentate ligament at all exposed levels [figures 1b c]. The thickened arachnoid tissue around the nerve roots in particular was dissected as far as possible to mobilize the spinal cord . The thickened arachnoid tissue was removed or resected, not in an en bloc fashion but fragment by fragment, so that some fragments of the thickened arachnoid tissue were left behind on the dorsal surface of the spinal cord . Next, a ventral dissection was started from both sides, with avoidance of excessive manipulation of the spinal cord [figures 1d e]. The ventral dissection was finally accomplished and pulsatile movement of the spinal cord could be visualized [figure 1f]. In all cases, a small rubber sheet was introduced on one side, passed below the spinal cord, and removed from the other side to ensure that the spinal cord was completely mobilized . After the dura mater and spinal cord were free from adhesions, an expansive dural plasty was performed using autologous fascia lata [figure 2a], followed by an expansive laminoplasty of the lift - up style [figure 2b]. Intraoperative photographs showing the surgical steps of the microsurgical arachnoidolysis in the procedure in case 1 . (b) higher magnifi cation of the boxed area in a revealed the thickened arachnoid tissue on the dorsolateral side of the spinal cord . (d) and (e) a ventral dissection was started from both sides with avoidance of excessive manipulation of the spinal cord . (f) the pulsative movement of the spinal cord was visualized at the fi nal step . Intraoperative photographs showing the dural plasty using autologous fascia lata (a), which was followed by expansive laminoplasty of lift - up style using hydroxyapatite laminar spacers (b). All four patients presented with initial symptoms of sensory disturbance, such as dysesthesia (three patients) or hypalgesia (one patient). The interval from the initial onset to surgery ranged from 2 to 18 years (mean, 9.5 years). The presumed etiologies of spinal adhesive arachnoiditis were infection in one patient and trauma in three patients . A clinical summary of the patients with focal adhesive arachnoiditis of the spinal cord is presented in table 1 . The focal adhesive lesion was assessed using ciss image or myelographic mr imaging with truefisp [figure 3]. In all patients, a focal adhesion could be discerned at a cervical or thoracic level [table 1]. Two patients showed modest or minor improvement in neurological function (cases 1 and 3), while two patients (cases 2 and 4) were unchanged, although the extent of the syringomyelia or myelomalacia was clearly decreased soon after surgery in all cases [figure 4]. Surgical outcome of the patients with focal adhesive arachnoiditis of spinal cord in case 2, serial myelographic mr imaging with truefisp at the level of the old spinal cord injury obtained before surgery suggests that the adhesive arachnoiditis was restricted to the injury site . Case 3: a 36-year - old male had been involved in a high - speed motor vehicle collision 2 years before . He was referred to our institute because of the gradual onset of sensory loss in the upper extremities, which had developed gradually after the traffic accident . Neurological examination on admission revealed severe hypalgesia in both upper extremities and complete paralysis below the injury level . Mri revealed the old damage of the spinal cord at t5, and diffuse extension of syringomyelia up to c4 [figure 4e]. Axial ciss images suggested the presence of focal adhesions on the ventral side of the spinal cord at the level of the old spinal cord injury, as the subarachnoid space on the ventral side was absent, with distortion of the spinal cord itself [figure 5]. The patient underwent microsurgical arachnoid dissection to mobilize the spinal cord and reconstruct the csf pathway . Following surgery, gradual improvement of sensory function in both upper extremities mri obtained soon after surgery revealed complete disappearance of the syringomyelia [figure 4f] and satisfactory expansion of the dural sac at the local site . During the follow - up period of 20 months, there was no neurological or radiological recurrence . T2-weighted mri obtained before surgery (a, c, e, and g) and late after surgery (b, d, f, and h) in all cases . A and b: case 1; c and d: case 2; e and f: case 3; g and h: case 4 . In case 3, serial axial ciss imaging at t4 (a), t5 (b), and t6 (c) obtained before surgery suggests that the focal adhesion is on the ventral side of spinal cord at the level of the old spinal cord injury (t5). Arachnoiditis can cause progressive syringomyelia or myelomalacia due to a tethering effect on the spinal cord, with consequent altered csf dynamics. [3461119] basic studies suggest that spinal tissue scarring at the injury site may cause a tethering effect on the spinal cord, which may lead to significant alterations in the spinal cord parenchyma . It is speculated that the rostrocaudal csf pulse wave is misdirected into the spinal cord parenchyma . The resulting high intramural pressure and decreased compliance of the subarachnoid space favor the flow of fluid into the spinal cord, possibly through perivascular spaces, resulting in a destructive cavitation process and eventually the formation of a syrinx cavity. [52023] causes of altered csf dynamics in the spinal subarachnoid space include adhesive arachnoiditis secondary to hemorrhage, infection, trauma, radiation necrosis, ischemic infarction, or surgery . In the present case series, the surgical procedure of microsurgical arachnoidolysis was indicated only when focal adhesive arachnoiditis was detected on mri using ciss imaging or myelographic mr imaging with truefisp . Ciss imaging is helpful to identify the neural structures within the subarachnoid space and is the sequence of choice for the preoperative diagnosis of trigeminal neuralgia, aqueductal stenosis, or brachial plexus lesions. [162527] myelographic mri with truefisp was first reported in 2003 by baskaran et al . And can provide superior assessment of spinal anatomy and abnormalities . In the evaluation of syringomyelia, unlike conventional t2-weighted mr imaging, ciss imaging and myelographic mri with truefisp can detect nerve root details, neural tethering, subarachnoid adhesions, or cavitation in the syrinx, allowing a significantly higher rate of detection of focal adhesive arachnoiditis and enabling the surgeon to accurately identify cases that require surgical treatment for obstructed csf . In the surgical treatment of syringomyelia or myelomalacia caused by adhesive arachnoiditis of the spinal cord, several approaches, such as shunting or microsurgical decompression of the subarachnoid space, have been reported. [12467111517192931] csf shunting procedures such as syringopleural or syringoperitoneal shunting have been increasingly used. [14] however, csf shunting procedures have shown an unsatisfactory prognosis and high rates of recurrence. [46] reported failure and complication rates of csf shunting procedures have reached 50%97%. [47] sgouros and williams concluded that only 50%53% of patients remained clinically stable after an average follow - up of 10 years . Reported that the causes of shunt malfunction were ingrowth of glial tissue into the perforations of the tubing, multiple separate syrinx cysts, tethering of the spinal cord by the catheter itself, infection, and low - pressure csf dynamics . Dissection of adhesive scarring of the spinal arachnoid membrane has been performed with microsurgical techniques aimed at obtaining free csf flow and untethering of the spinal cord . Reported their multicenter experience with treatment of 78 patients with spinal syringomyelia associated with posttraumatic or postinflammatory arachnoid scarring; the mean follow - up time was 32 months . Of 121 procedures, 64 were syrinx shunt placement procedures and the authors report long - term recurrence rates of 97%, generally with clinical recurrence within 2 years . They advocate careful microsurgical dissection of the arachnoid scar and decompression of the subarachnoid space with a dural graft . In their experience, this yielded clinical stabilization of preoperative progressive neurological deterioration in 83% of patients . Microsurgical arachnoidolysis appears to be a straightforward method for mobilizing the spinal cord and reconstructing the csf pathway, though the surgical procedure is technically demanding and challenging . It is conceivable that microsurgical arachnoid dissection can be used in focal arachnoiditis with possible favorable outcomes on imaging as well as in terms of neurological function, although surgery of longitudinally extensive arachnoiditis needs to be further explored . First, cutting the dentate ligament and the arachnoid membrane rostral and caudal to the lesion is desirable before starting meticulous arachnoidolysis of the adhesions . Second, the dorsal surface of the spinal cord around the lesion should never be dissected because the spinal cord segment around the lesion may be fragile and easily damaged by the surgical manipulation . Third, the spinal cord should be mobilized as much as possible, with restoration of the csf pathway . Finally, the surgical field should be clean and bloodless to avoid retethering of the spinal cord long after the microsurgical arachnoidolysis . The csf pathway can be maintained by a duraplasty using fascia lata, followed by expansive laminoplasty . The material used for duraplasty is another important factor that determines a long - lasting benefit . Although a gore - tex membrane has been used to prevent or minimize postoperative readhesion, the neural tissue was bound to a thin neomembrane that surrounded the gore - tex membrane in an experimental study in rats . Dural plasty using autologous fascia lata may be better for reducing the risk of adhesive change long after the surgery . In addition, dural plasty should be stretched and expanded by securing it to the adjacent bone, to maintain the csf pathway . Ciss imaging or myelographic mr imaging with truefisp is helpful for localizing the lesion for surgical exposure . The surgical outcomes in the cases presented are preliminary, but acceptable in the follow - up period . We conclude that microsurgical arachnoidolysis appears to be a straightforward method for stabilizing the progressive symptoms, although the surgical procedure is technically demanding.
Type 2 diabetes mellitus (t2 dm) is a progressive disease, meaning that most patients will require the introduction of insulin into their treatment regimen at some point during the continuum of care 1 . To facilitate the transition to insulin, numerous clinical and real - world studies have investigated the efficacy and safety of initiating long - acting analogue insulins . In clinical studies, both insulin glargine (gla; lantus, sanofi us, inc ., bridgewater, nj, usa) and insulin detemir (det; levemir, novo nordisk a / s, bagsvaerd, denmark) have been shown to result in equivalent improvements in glycaemic control with a low incidence of hypoglycaemia when used as part of a basal - bolus regimen 24 . Although glycaemic outcomes in such trials have been similar for these two basal analogue insulins, it has been noted that higher doses and twice - daily dosing are often required with det compared with gla 3,4 . A recent trial has suggested that when both det and gla are used once daily as an adjunct to metformin, a greater proportion of patients treated with gla reached glycated haemoglobin (hba1c) levels <7.0% than did patients treated with det 5 . Attempts to examine the relative advantages of these insulin analogues in a real - world setting have proved difficult to interpret . Some studies have reported enhanced glycaemic control with gla compared with det, along with improved adherence and persistence, with no difference with regard to hypoglycaemia, healthcare costs or weight gain 68; however, other studies found no difference in glycaemic control between the two insulin types 9,11, with one suggesting that patients initiating det were 30% less likely to gain 0.9 kg or more in body weight than gla users 10 . Studies investigating the efficacy and safety of switching from gla to det are similarly conflicting in their findings . Although once - daily dosing of gla and det has been shown to result in equivalent 24-h glycaemic control, switching from gla to det was associated with improved hba1c levels and fewer hypoglycaemic events, compared with remaining on gla in an observational study of patients with type 1 diabetes mellitus (t1 dm) and t2 dm 7,11,12 . A retrospective analysis, however, reported that switching from gla to det did not improve glycaemic control among a cohort of patients with t2 dm 13 . A recent longitudinal study sought to expand on these data by assessing real - world outcomes using a retrospective analysis of two large, independent, national, us databases comprising commercially insured and medicare populations: the impact and humana cohorts 14 the study found that, at 1-year follow - up, patients who had remained on gla showed significantly higher persistence with and adherence to treatment compared with those who switched to det (p <0.001). In addition, 37% of patients who switched to det restarted gla (p <0.05), compared with only 20% of gla users returning to det after having switched to gla . Overall hypoglycaemia rates were significantly higher for patients continuing on gla than for patients switching to det in the humana cohort (16 vs. 11%; p <0.05), but overall hypoglycaemia rates were similar in the impact cohort (11 vs. 12%; p = 0.490). In the present study, to further evaluate the consequences of insulin switching, we used data from patients' medical records to investigate retrospectively the real - world clinical outcomes for patients with t2 dm who switched between these two basal insulin analogues . The present analysis was a retrospective cohort study using the ge centricity electronic medical records (emr) database from 1 july 2005 to 31 december 2012 . In 2007, the ge centricity emr database contained the medical records of 30 million patients in 49 us states . As the analysis was performed on de - indentified data, approval from an ethics committee was not necessary . We analysed two cohorts in the present study: cohort 1 included patients who were previously treated with gla and switched to det (det - s) or who remained on gla (gla - c); and cohort 2 included patients previously treated with det and subsequently switched to gla (gla - s) or who remained on det (det - c). In cohort 1, the patients in the det - s subgroup were required to have 1 physician prescription for gla <6 months before first det physician prescription order date (after 1 january 2006). Patients in the gla - c subgroup were required to have 2 gla physician prescriptions starting from the third - quarter of 2005, without a subsequent physician prescription order of det, with 1 of the orders being the index date was a randomly selected date between the second to the last gla physician prescription . In cohort 2, patients in the gla - s subgroup were required to have 1 physician prescription for det <6 months before the first gla prescription order date (after 1 july 2006). Patients in the det - c subgroup were required to have 2 det physician prescriptions starting from the first - quarter of 2006, without a subsequent physician prescription order for gla . The index date was a randomly selected date between the second and last det physician prescriptions dates . Patients aged 18 years on the index date were included in the analysis if they had a diagnosis of t2 dm [international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes: 250.x0 or 250.x2] with emr activity data for 6 months pre - index (baseline) and 12 months post - index (follow - up). Patients were further required to have: 1 baseline hba1c measure taken during the period 90 days before up to 15 days after the index date; 1 baseline body mass index (bmi) measure taken in the period 30 days before up to 15 days after the index date; and 1 follow - up hba1c measure from the second - quarter until 3 months after the 1-year follow - up . In cohort 1, patients with baseline prescriptions for premixed insulin or basal insulins other than gla [i.e. Neutral protamine hagedorn (nph) insulin, lente, or ultralente] were excluded . Similarly, in cohort 2, patients with baseline prescriptions for premixed insulin or basal insulins other than det (i.e. Nph insulin, lente or ultralente) were excluded . Baseline demographic and clinical characteristics included age, gender, region, race, geographic region, insurance type, bmi, weight, individual comorbidities and prescribed diabetes medications . In addition, the charlson comorbidity index (cci) score was calculated, which is the weighted sum of 19 categories of comorbidity defined using icd-9-cm diagnoses codes, with a higher score indicating a more severe burden of comorbidity and a higher mortality risk 15,16 . Clinical outcome variables were measured 1 year after the index date (follow - up) and included follow - up hba1c, change in hba1c from baseline, follow - up hypoglycaemic events, follow - up bmi, change in bmi from baseline follow - up body weight, change in body weight from baseline and follow - up oral antidiabetes drug (oad) use (including gla, det and rapid - acting insulin). Baseline hba1c measures were defined as those occurring between 90 days before and up to 15 days after the index date; baseline body weight and bmi measures were defined as 1 baseline measurement between 30 days before and up to 15 days after the index date . Follow - up hba1c values were defined as those occurring during the period from the second - quarter until 3 months after 1-year follow - up, and follow - up body weight and bmi values were defined as those occurring during the 30 days before or after the end of the 1-year follow - up period (index date + 360 days). If patients had multiple hba1c, body weight or bmi results during this period, the value dated closest to the index date was used as the baseline value, and the value dated closest to the end of the follow - up period was used as the follow - up value . Hypoglycaemia was defined as a healthcare encounter (outpatient, inpatient or emergency department visit) with a primary or secondary icd-9-cm diagnosis code for hypoglycaemia, based on the algorithm published by ginde et al . 17 . Both the proportion of patients with 1 hypoglycaemic event (prevalence rate) and the average number of hypoglycaemic events per patient (event rate) during the baseline and 1-year follow - up period were identified . Treatment persistence was defined as the patient remaining on the index long - acting insulin (gla for gla - c and det for det - s for cohort 1; det for det - c and gla for gla - s for cohort 2) during the 1-year follow - up period without discontinuation after study drug initiation . Based on the published methodology 18, study medication was considered discontinued if the prescription was not re - ordered by the physician within the expected time of medication coverage (the pre - defined 90th percentile of the time between first and second physician prescriptions among patients with 1 prescription). Patients who restarted their initial medication after a period of not having used this medication during follow - up were considered non - persistent . Persistence days were measured as the number of days between the index date and the discontinuation date . Sensitivity analyses were also conducted using 75th and 95th percentiles of the expected time of medication coverage . Descriptive and multivariate statistical methodologies were used to assess the baseline characteristics and to compare the outcomes over the 1-year follow - up period . To address potential selection bias in the real - world setting, in each cohort, patients from the two subgroups were matched by propensity - score matching, with a 1 up to 5 ratio in cohort 1 and a 1 up to 3 ratio in cohort 2, to balance baseline age, gender, race, geographic region, insurance type, physician specialty, index year, baseline comorbidity, baseline oad use, concomitant medication, hba1c and bmi . These ratios and matchings were chosen because our preliminary analysis revealed a prescription imbalance, with significantly more eligible patients in the gla group when compared with the det group, and a much lower number of patients in the additionally, one - to - many matching has previously been validated as a method to increase precision in cohort studies, compared with one - to - one matching, and has also been supported in a recent review assessing the quality of statistical methodologies in matched case matching was implemented without replacement and any patient without 1 match was excluded from the analysis . Between - group covariate balance was evaluated using descriptive t - tests and chi - squared tests (with corresponding p - values) and standardized differences, where a standardized difference <10 indicated adequate balance 21 . Because not every patient in the matched cohorts had follow - up bmi or body weight data, we conducted, as sensitivity analysis, ordinary least squares regressions for bmi and body weight changes, adjusting for potential baseline imbalances . The model used a larger set of covariates, which included patient demographics (age, gender, geographic locations and bmi categories), baseline comorbidities and some basic clinical characteristics (baseline oad use, statins for baseline concomitant medication use, baseline hba1c level and baseline hypoglycaemic events). We analysed two cohorts in the present study: cohort 1 included patients who were previously treated with gla and switched to det (det - s) or who remained on gla (gla - c); and cohort 2 included patients previously treated with det and subsequently switched to gla (gla - s) or who remained on det (det - c). In cohort 1, the patients in the det - s subgroup were required to have 1 physician prescription for gla <6 months before first det physician prescription order date (after 1 january 2006). Patients in the gla - c subgroup were required to have 2 gla physician prescriptions starting from the third - quarter of 2005, without a subsequent physician prescription order of det, with 1 of the orders being the index date was a randomly selected date between the second to the last gla physician prescription . In cohort 2, patients in the gla - s subgroup were required to have 1 physician prescription for det <6 months before the first gla prescription order date (after 1 july 2006). Patients in the det - c subgroup were required to have 2 det physician prescriptions starting from the first - quarter of 2006, without a subsequent physician prescription order for gla . The index date was a randomly selected date between the second and last det physician prescriptions dates . Patients aged 18 years on the index date were included in the analysis if they had a diagnosis of t2 dm [international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes: 250.x0 or 250.x2] with emr activity data for 6 months pre - index (baseline) and 12 months post - index (follow - up). Patients were further required to have: 1 baseline hba1c measure taken during the period 90 days before up to 15 days after the index date; 1 baseline body mass index (bmi) measure taken in the period 30 days before up to 15 days after the index date; and 1 follow - up hba1c measure from the second - quarter until 3 months after the 1-year follow - up . In cohort 1, patients with baseline prescriptions for premixed insulin or basal insulins other than gla [i.e. Neutral protamine hagedorn (nph) insulin, lente, or ultralente] were excluded . Similarly, in cohort 2, patients with baseline prescriptions for premixed insulin or basal insulins other than det (i.e. Nph insulin, lente or ultralente) were excluded . Baseline demographic and clinical characteristics included age, gender, region, race, geographic region, insurance type, bmi, weight, individual comorbidities and prescribed diabetes medications . In addition, the charlson comorbidity index (cci) score was calculated, which is the weighted sum of 19 categories of comorbidity defined using icd-9-cm diagnoses codes, with a higher score indicating a more severe burden of comorbidity and a higher mortality risk 15,16 . Clinical outcome variables were measured 1 year after the index date (follow - up) and included follow - up hba1c, change in hba1c from baseline, follow - up hypoglycaemic events, follow - up bmi, change in bmi from baseline follow - up body weight, change in body weight from baseline and follow - up oral antidiabetes drug (oad) use (including gla, det and rapid - acting insulin). Baseline hba1c measures were defined as those occurring between 90 days before and up to 15 days after the index date; baseline body weight and bmi measures were defined as 1 baseline measurement between 30 days before and up to 15 days after the index date . Follow - up hba1c values were defined as those occurring during the period from the second - quarter until 3 months after 1-year follow - up, and follow - up body weight and bmi values were defined as those occurring during the 30 days before or after the end of the 1-year follow - up period (index date + 360 days). If patients had multiple hba1c, body weight or bmi results during this period, the value dated closest to the index date was used as the baseline value, and the value dated closest to the end of the follow - up period was used as the follow - up value . Hypoglycaemia was defined as a healthcare encounter (outpatient, inpatient or emergency department visit) with a primary or secondary icd-9-cm diagnosis code for hypoglycaemia, based on the algorithm published by ginde et al . 17 . Both the proportion of patients with 1 hypoglycaemic event (prevalence rate) and the average number of hypoglycaemic events per patient (event rate) during the baseline and 1-year follow - up period were identified . Treatment persistence was defined as the patient remaining on the index long - acting insulin (gla for gla - c and det for det - s for cohort 1; det for det - c and gla for gla - s for cohort 2) during the 1-year follow - up period without discontinuation after study drug initiation . Based on the published methodology 18, study medication was considered discontinued if the prescription was not re - ordered by the physician within the expected time of medication coverage (the pre - defined 90th percentile of the time between first and second physician prescriptions among patients with 1 prescription). Patients who restarted their initial medication after a period of not having used this medication during follow - up were considered non - persistent . Persistence days were measured as the number of days between the index date and the discontinuation date . Sensitivity analyses were also conducted using 75th and 95th percentiles of the expected time of medication coverage . Descriptive and multivariate statistical methodologies were used to assess the baseline characteristics and to compare the outcomes over the 1-year follow - up period . To address potential selection bias in the real - world setting, in each cohort, patients from the two subgroups were matched by propensity - score matching, with a 1 up to 5 ratio in cohort 1 and a 1 up to 3 ratio in cohort 2, to balance baseline age, gender, race, geographic region, insurance type, physician specialty, index year, baseline comorbidity, baseline oad use, concomitant medication, hba1c and bmi . These ratios and matchings were chosen because our preliminary analysis revealed a prescription imbalance, with significantly more eligible patients in the gla group when compared with the det group, and a much lower number of patients in the additionally, one - to - many matching has previously been validated as a method to increase precision in cohort studies, compared with one - to - one matching, and has also been supported in a recent review assessing the quality of statistical methodologies in matched case matching was implemented without replacement and any patient without 1 match was excluded from the analysis . Between - group covariate balance was evaluated using descriptive t - tests and chi - squared tests (with corresponding p - values) and standardized differences, where a standardized difference <10 indicated adequate balance 21 . Because not every patient in the matched cohorts had follow - up bmi or body weight data, we conducted, as sensitivity analysis, ordinary least squares regressions for bmi and body weight changes, adjusting for potential baseline imbalances . The model used a larger set of covariates, which included patient demographics (age, gender, geographic locations and bmi categories), baseline comorbidities and some basic clinical characteristics (baseline oad use, statins for baseline concomitant medication use, baseline hba1c level and baseline hypoglycaemic events). A total of 25 290 eligible patients were identified with 19 811 patients from cohort 1 (gla - c, n = 17 783 and det - s, n = 2028) and 5479 from cohort 2 (det - c, n = 4431 and gla - s, n = 1048). Significant baseline differences existed between the subgroups in each cohort (table s1). In cohort 1, when compared with patients in the det - s subgroup, patients in the gla - c subgroup were older, were more likely to be black, were more likely to reside in the midwest or west, were more likely to be enrolled in medicare, had higher cci scores and had lower hba1c values . No significant differences were found for bmi or body weight between the gla - c and det - s subgroups at baseline . In cohort 2, when compared with the det - c subgroup, patients in the gla - s subgroup were younger, were more likely to be black, were more likely to reside in the midwest or west, had higher hba1c values, and had higher body weight and bmi values . Patients in the gla - s and det - c subgroups had similar cci scores at baseline . For both cohorts, no significant differences were found for baseline hypoglycaemic events between groups . Bmi, body mass index; emr, electronic medical records; hba1c, glycated haemoglobin; t2 dm, type 2 diabetes mellitus . After propensity - score matching, the final matched study population included 13 942 patients overall, with 10 657 from cohort 1 (gla - c, n = 8860 and det - s, n = 1797; female patients 54.7%, age 58.4 years, hba1c level 8.7%, bmi 35.1 kg / cm, weight 99.3 kg) and 3285 patients from cohort 2 (det - c, n = 2427 and gla - s, n = 858; female 53.5%, age 58.3 years, hba1c level 8.8%, bmi 35.4 kg / cm, weight 100.7 kg). The baseline demographic and clinical characteristics were balanced between treatment groups in each cohort (table1). Baseline characteristics among matched patients in cohorts 1 and 2 det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det; bmi, body mass index; cci, charlson comorbidity index; hba1c, glycated haemoglobin; oad, oral antidiabetes drug; s.d ., standard deviation . During 1-year follow - up, overall, switching from gla to det resulted in lower persistence with treatment, whereas switching from det to gla resulted in higher persistence . Among patients in cohort 1, those in the det - s subgroup versus those in the gla - c subgroup had significantly lower persistence with treatment (52.4 vs. 61.3%, respectively; p <0.0001) and significantly fewer treatment persistent days (311.6 vs. 331.0 days; p <0.0001). In cohort 2, however, the gla - s and the det - c subgroups had similar levels of treatment persistence (56.5 vs. 58.5%, respectively; p = 0.292) yet fewer treatment persistent days (320.7 vs. 325.3 days; p = 0.015). Sensitivity analysis using 75th and 95th percentile of the time duration yielded similar results: 75th percentile: cohort 1 gla - c versus det - s subgroup, 21.2 versus 35.7% (p <0.0001); cohort 2 det - c versus gla - s subgroup, 24.7 versus 31.6% (p = 0.0001); 95th percentile: cohort 1 gla - c versus det - s subgroup, 63.1 versus 68.2% (p <0.0001); cohort 2 det - c versus gla - s subgroup, 67.0 versus 66.0% (p = 0.622). In cohort 1, the use of rapid - acting insulin was significantly higher among the patients in the det - s subgroup than among those in the gla - c subgroup (57.6 vs. 52.5%; p <0.0001; figure 2), and 26.9% of patients in the det - s subgroup restarted gla . In cohort 2, however, similar proportions of patients used rapid - acting insulin in the det - c and gla - s subgroups (50.4 vs. 51.1%; p = 0.712), and 18.6% of patients in the gla - s subgroup restarted det . Patients requiring rapid - acting insulin (rai). Det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det . The baseline mean hba1c level was similar between the treatment groups in each of the cohorts . In cohort 1, patients in the det - s subgroup had a significantly higher follow - up hba1c level (figure 3a), a significantly lower hba1c reduction (figure 3b), and significantly lower proportions of patients achieved hba1c <7.0% (figure 3c) and hba1c <8.0% (figure 3d) at the end of 1-year follow - up compared with patients in the gla - c subgroup . In cohort 2, however, compared with patients in the det - c subgroup, patients in the gla - s subgroup had a significantly lower follow - up hba1c (figure 3a) and a numerically higher (although not statistically significant) hba1c reduction (figure 3b), but there were no differences in the proportions of patients achieving an hba1c level <7.0% (figure 3c) or <8.0% (figure 3d). (a) one - year follow - up glycated haemoglobin (hba1c) values, (b) change in hba1c from baseline, (c) proportion of patients achieving target hba1c <7.0%, and (d) proportion of patients achieving target hba1c <8.0% . Det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det . For both prevalence and event rates of any hypoglycaemic events, no significant differences were found after follow - up between the det - s and gla - c subgroups in cohort 1 or between the gla - s and det - c subgroups in cohort 2 [prevalence rate: 2.0 vs. 2.1%, p = 0.889; event rate (number of events / patient year): 0.023 vs. 0.026, p = 0.4424] or between the gla - c and det - s subgroups in cohort 2 (2.3 vs. 1.6%, p = 0.2314; event rate: 0.028 vs. 0.021, p = 0.3727). In both cohorts, baseline bmi and body weight were similar between the groups (table1). In cohort 1, bmi at 1-year follow - up was 35.0 kg / m for both the det - s and gla - c subgroups (p = 0.843). There were also no significant differences in bmi change for patients in the det - s and gla - c subgroups after follow - up (figure 4a). The 1-year follow - up weight was similar for the det - s and gla - c subgroups (99.3 vs. 99.4 kg; p = 0.881), with similar weight changes between patients in the det - s subgroup and those in the gla - c subgroup (figure 4b). Changes in (a) body mass index (bmi) and (b) body weight at 1-year follow - up . Det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det . In cohort 2, at the end of 1-year follow - up, patients in the gla - s subgroup had a higher bmi (36.3 vs. 35.5 kg / m; p = 0.052), but this was not statistically significant . There were no significant differences at 1-year follow - up for body weight between patients in the gla - s and det - c subgroups (102.2 vs. 100.9 kg; p = 0.299); however, patients in the gla - s subgroup had a greater change from baseline in body weight compared with those in the det - c subgroup (figure 4b). Sensitivity analyses using ordinary least squares regressions yielded similar results: adjusted weight change: cohort 1 det - s versus gla - c subgroup, 0.18 versus + 0.10 kg (p = 0.329); cohort 2 gla - s versus det - c subgroup, + 1.48 versus + 0.02 kg (p <0.001); adjusted bmi change: cohort 1 gla - c versus det - s subgroup, 0.18 versus 0.04 kg (p = 0.122); cohort 2 gla - s versus det - c subgroup, + 0.48 versus 0.05 kg (p <0.001). During 1-year follow - up, overall, switching from gla to det resulted in lower persistence with treatment, whereas switching from det to gla resulted in higher persistence . Among patients in cohort 1, those in the det - s subgroup versus those in the gla - c subgroup had significantly lower persistence with treatment (52.4 vs. 61.3%, respectively; p <0.0001) and significantly fewer treatment persistent days (311.6 vs. 331.0 days; p <0.0001). In cohort 2, however, the gla - s and the det - c subgroups had similar levels of treatment persistence (56.5 vs. 58.5%, respectively; p = 0.292) yet fewer treatment persistent days (320.7 vs. 325.3 days; p = 0.015). Sensitivity analysis using 75th and 95th percentile of the time duration yielded similar results: 75th percentile: cohort 1 gla - c versus det - s subgroup, 21.2 versus 35.7% (p <0.0001); cohort 2 det - c versus gla - s subgroup, 24.7 versus 31.6% (p = 0.0001); 95th percentile: cohort 1 gla - c versus det - s subgroup, 63.1 versus 68.2% (p <0.0001); cohort 2 det - c versus gla - s subgroup, 67.0 versus 66.0% (p = 0.622). In cohort 1, the use of rapid - acting insulin was significantly higher among the patients in the det - s subgroup than among those in the gla - c subgroup (57.6 vs. 52.5%; p <0.0001; figure 2), and 26.9% of patients in the det - s subgroup restarted gla . In cohort 2, however, similar proportions of patients used rapid - acting insulin in the det - c and gla - s subgroups (50.4 vs. 51.1%; p = 0.712), and 18.6% of patients in the gla - s subgroup restarted det . Det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det . The baseline mean hba1c level was similar between the treatment groups in each of the cohorts . In cohort 1, patients in the det - s subgroup had a significantly higher follow - up hba1c level (figure 3a), a significantly lower hba1c reduction (figure 3b), and significantly lower proportions of patients achieved hba1c <7.0% (figure 3c) and hba1c <8.0% (figure 3d) at the end of 1-year follow - up compared with patients in the gla - c subgroup . In cohort 2, however, compared with patients in the det - c subgroup, patients in the gla - s subgroup had a significantly lower follow - up hba1c (figure 3a) and a numerically higher (although not statistically significant) hba1c reduction (figure 3b), but there were no differences in the proportions of patients achieving an hba1c level <7.0% (figure 3c) or <8.0% (figure 3d). (a) one - year follow - up glycated haemoglobin (hba1c) values, (b) change in hba1c from baseline, (c) proportion of patients achieving target hba1c <7.0%, and (d) proportion of patients achieving target hba1c <8.0% . Det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det . For both prevalence and event rates of any hypoglycaemic events, no significant differences were found after follow - up between the det - s and gla - c subgroups in cohort 1 or between the gla - s and det - c subgroups in cohort 2 [prevalence rate: 2.0 vs. 2.1%, p = 0.889; event rate (number of events / patient year): 0.023 vs. 0.026, p = 0.4424] or between the gla - c and det - s subgroups in cohort 2 (2.3 vs. 1.6%, p = 0.2314; event rate: 0.028 vs. 0.021, p = 0.3727). In both cohorts, baseline bmi and body weight were similar between the groups (table1). In cohort 1, bmi at 1-year follow - up was 35.0 kg / m for both the det - s and gla - c subgroups (p = 0.843). There were also no significant differences in bmi change for patients in the det - s and gla - c subgroups after follow - up (figure 4a). The 1-year follow - up weight was similar for the det - s and gla - c subgroups (99.3 vs. 99.4 kg; p = 0.881), with similar weight changes between patients in the det - s subgroup and those in the gla - c subgroup (figure 4b). Changes in (a) body mass index (bmi) and (b) body weight at 1-year follow - up . Det, insulin detemir; gla, insulin glargine; det - s, patients previously treated with gla who switched to det; gla - c, patients who remained on gla; gla - s, patients previously treated with det who switched to gla; det - c, patients who remained on det . In cohort 2, at the end of 1-year follow - up, patients in the gla - s subgroup had a higher bmi (36.3 vs. 35.5 kg / m; p = 0.052), but this was not statistically significant . Change in bmi was significantly different (figure 4a). There were no significant differences at 1-year follow - up for body weight between patients in the gla - s and det - c subgroups (102.2 vs. 100.9 kg; p = 0.299); however, patients in the gla - s subgroup had a greater change from baseline in body weight compared with those in the det - c subgroup (figure 4b). Sensitivity analyses using ordinary least squares regressions yielded similar results: adjusted weight change: cohort 1 det - s versus gla - c subgroup, 0.18 versus + 0.10 kg (p = 0.329); cohort 2 gla - s versus det - c subgroup, + 1.48 versus + 0.02 kg (p <0.001); adjusted bmi change: cohort 1 gla - c versus det - s subgroup, 0.18 versus 0.04 kg (p = 0.122); cohort 2 gla - s versus det - c subgroup, + 0.48 versus 0.05 kg (p <0.001). The aim of the present study was to evaluate retrospectively the real - world clinical outcomes of switching between basal insulin analogues, compared with continuing on previous basal insulin, among patients with t2 dm identified from the us ge centricity emr database . Overall, contrasting findings were observed when patients switched from gla to det, as compared with switching from det to gla . In cohort 1, compared with patients in the det - s subgroup, patients in the gla - c subgroup had better clinical outcomes at 1-year follow - up, including lower hba1c levels and significantly higher hba1c reductions . In addition, significantly more patients in the gla - c subgroup achieved the targeted hba1c levels of <7.0% or <8.0% and those in the det - s subgroup had significantly higher rapid - acting insulin use . In addition, no significant differences in hypoglycaemia rates and weight / bmi were observed between the two groups . These results could be attributable to lower persistence / adherence as patients might not be taking their full medication . In cohort 2, however, patients in the gla - s subgroup had lower hba1c levels and a numerically higher (but non - significant) hba1c reduction; similar proportions of patients achieved hba1c levels of <7.0 or <8.0%, and similar hypoglycaemia rates were seen in the two groups . Although 1-year follow - up body weight and bmi were similar, patients in the det - c subgroup had a decreased bmi and a lower body weight increase, whereas patients in the gla - s subgroup had increased bmi and significantly greater body weight increase . Although the patients in the gla - s subgroup gained weight, the increase was <1% which, it is suggested, is not clinically relevant 22, and may reflect differences in follow - up plasma glucose levels or other factors . Furthermore, the increase in weight with gla could be taken to be a sign of better compliance . If a switch between insulin analogues is of a disruptive nature, it will result in decreased compliance . For example, if a regimen becomes more complex (from once daily to twice daily, adding a rapid - acting insulin etc . ), it could lead to less of the typical weight gain associated with insulin treatment . In cohort 1, the patients in the det - s subgroup, who switched from gla to det, had lower persistence than those who remained on gla (robust in sensitivity analyses). This could be attributable to the fact that most patients on det administer twice daily, whereas most patients on gla administer once daily 13,23 . Unfortunately, this information is not captured in the ge centricity emr database . By contrast, in cohort 2, data on patients in the gla - s subgroup switching from det to gla suggested similar overall persistence with treatment . Approximately 27% of the patients in the det - s subgroup restarted gla during the follow - up period . Of patients in the gla - s subgroup although the reasons for restarting the former insulin are not captured in the ge centricity emr database, these findings are consistent with a previous impact and humana database study 14 . Unlike randomized controlled trials, where patients are required to be persistent with their therapy, real - world patients and physicians do not follow a stringent protocol and, therefore, any differences in patients' persistence with their insulin treatment can translate into differences in clinical and economic outcomes 17 . Overall, contrasting outcomes were observed when patients were switched from gla to det, when compared with switching from det to gla . This raises questions about the therapeutic interchangeability of these two basal analogue insulins in clinical practice, although further studies are required to confirm this non - interchangeability . The results of the present ge centricity emr switching analysis, however, were highly consistent with the previously performed claims database analysis 14 . The ge centricity emr database is a large and comprehensive source of data to understand clinical practice and outcomes in the real - world setting . This database contains rich clinical information, such as hba1c level, weight and bmi, that is not available in insurance claim databases . Overall 25 000 patients were identified in this study, and almost 15 000 were propensity - score - matched and analysed, including those switching from det to gla and those switching from gla to det during the same time period . In previous observational studies on switching from gla to det 11,13,24, conflicting results were shown with relatively low numbers of patients included (301300 patients) and including either only patients with t1 dm, or a mixed group of patients with t1 dm and t2 dm . Our previous study used administrative claims data 14, and the results described in the present study not only validate our previous findings with a different data source but also strengthen them with additional data on weight . One of the key limitations of the present study, however, is that the emr analysis was carried out on primary care physician prescription data, rather than on claims databases . Prescription orders do not necessarily mean that the prescriptions were filled and taken as directed . In addition, certain data were not available, including glucose levels, the duration of treatment with the initial insulin, the duration of t2 dm, and the reason for switching insulin (e.g. Insurance required switching to other insulin). Although propensity - score matching was used to balance the baseline data between treatment groups, the differences between the groups were quite significant (table s1) and the potential residual confounding might therefore still be present, particularly unobserved selection bias . In a real - world setting, switching patients to another treatment regimen may introduce confounding by indication, an important limitation to observational studies reporting on patients changing their treatment regimens, including the study described here . Other potential sources of bias and confounding include the range of time from measurement of hba1c to index date, differences in time for follow - up hba1c and follow - up bmi, and patient / physician preference . In addition, the dose of insulin at the time of switching was not available in the ge centricity emr database and might have impacted on clinical outcomes, especially in patients who had recently switched insulin analogues and had not had the opportunity to titrate their dosage . Specifically, the present study was designed to compare between patients who switched to a different basal insulin and those who continued on the previous basal insulin . In the case of comparing patients in the det - s with those in the gla - c subgroup, patients in the latter subgroup may already be fully titrated, while it takes time for patients switching to det to titrate . This may lead to an overestimation of the benefits of gla compared with det, representing a further limitation of this analysis . The same limitation applies to the comparison of the gla - s with the det - c subgroup . One approach to account for confounding by indication is to compare patients in the gla - s subgroup with those in the det - s subgroup directly; however, these patients had different baseline doses of basal insulin, and based on a real - world study among us patients initiating gla or det, it may be that higher insulin doses were used in the det - treated patients 6 . It is not possible to establish causality from the differences in outcomes, nor are the data necessarily representative of all patients with t2 dm . Patients could drop out for various reasons and thus selection may occur . In the present study, more patients treated with det than those treated with gla dropped out from the study population (56 vs. 35%) when 6-month baseline and 12-month follow - up were required . Similarly, only 8% of patients on det treatment compared with 40% on gla, dropped out when being aged> 18 years was required (figure 1). Sufficient data on hba1c levels and bmi were missing for half of the patients in this analysis, and this lack of completeness should be considered as a major limitation of emr databases . When interpreting the findings of the present study, it should be remembered that, because of its limitations, the methodology used may have introduced bias or affected the because of the real - world nature of the present study, patients did not have their hba1c levels or bmi evaluated regularly and thus were excluded if they did not have baseline and/or follow - up hba1c / bmi values . Overall, recognizing the inherent limitations of real - world data analyses, the findings of the present study alone should not be used to guide clinicians in the decision to switch insulin, but the findings do merit further investigation . Future studies should look more closely at a number of other factors, including the dose, duration of treatment, reasons for and cost - effectiveness of switching in patients with t2 dm . In addition, although previous studies have suggested that in patients with t1 dm det was associated with a higher dose and more frequent twice - daily dosing than gla 13,24, large cohort comparative studies in patients with t1 dm should be conducted to confirm these findings . In conclusion, the results of the present study suggest that switching patients with t2 dm from gla to det might lead to decreases in treatment persistence alongside having a negative effect on glycaemic outcomes . Maintaining patients on gla or switching from det to gla, however, might be beneficial, although may result in minor weight gain . This suggests that the two basal insulin analogues might not be therapeutically interchangeable, and further studies are required to investigate this hypothesis . P. l. is a member of the advisory panel for sanofi us, inc . And, has received research support from eli lilly and co., sanofi us, inc ., novo nordisk, inc ., amylin pharmaceuticals, inc . And boehringer ingelheim pharmaceuticals, inc ., and serves on speaker's bureaus for eli lilly and co., novo nordisk, inc ., amylin pharmaceuticals, inc . And boehringer ingelheim pharmaceuticals, inc . W. w., r. m., f. y. and j. g. are employees of sanofi us, inc . L. x. and o. b. are employees of statinmed, which received funding to carry out this work from sanofi us, inc . P. l. co - developed the concept, interpreted the results of the analyses, reviewed manuscript outline and drafts and provided comments . W. w. proposed and co - developed the concept, co - developed the analysis plan, interpreted the results of the analyses, reviewed manuscript outline and drafts and provided comments . R. m. co - developed the concept, co - developed the analysis plan, interpreted the results of the analyses, reviewed manuscript outline and drafts and provided comments . F. y. collected the data, co - developed the analysis plan, conducted statistical analyses and interpreted the results, reviewed manuscript outline and drafts and provided comments . L. x. collected the data, co - developed the analysis plan, conducted statistical analyses and interpreted the results, reviewed manuscript outline and drafts and provided comments . O. b. interpreted the results of the analyses, reviewed manuscript outline and drafts and provided comments . J. g. developed the concept, co - developed the analysis plan, interpreted the results of the analyses, reviewed manuscript outline and drafts and provided comments . Additional supporting informationmay be found in the online version of this article: baseline characteristics among prematched patients of cohorts 1 and 2.
Ocular parasitosis in human is more prevalent in geographical areas where environmental factors and poor sanitary conditions favor the parasitism between man and animals . In recent years, population shift and rapid transport have facilitated the spread of certain parasitic diseases from endemic to nonendemic areas . The routes of infection to man vary with species of the parasite and the animal hosts they infest . Lesions in the eye can be due to damage directly caused by the infectious pathogen, indirect pathology caused by toxic products, immune response incited by infections, or ectopic parasitism of the preadult or adult stages . The epidemiology of parasitic ocular diseases reflects the habitat of the causative parasites as well as the habits and health status of the patient . Additional consideration must include local sanitation and the presence of a vector for transmission as well as the more complicated life cycles of the parasites and definitive hosts . Dietary history should be considered since most parasitic transmission is through food and water contamination . An awareness of these is therefore important to the clinician evaluating this group of patients . An ocular examination may provide clues to the underlying disease, and an awareness of the possibilities of travel - related pathology may shed light on an ocular presentation . The eye is involved both in a variety of systemic infections and may be the primary focus of other pathologies . The ophthalmic lesions occur as a result of antiparasitic treatment as it has been noticed in the prophylactic and therapeutic attempts to treat malaria [1, 2]. Drugs such as hydroxychloroquine and chloroquine can damage vision because of their toxic effects, which is due to slow accumulation of the drugs in the retinal epithelium that results in irreversible visual loss . Much debate and confusion have taken place over the type and frequency of ocular examination in patients taking these drugs . Despite improved understanding of the clinical features of inflammatory eye diseases and advances in diagnostic testing, clinicians should maintain a high index of suspicion for infective parasitic diseases in patients thought to have inflammatory eye involvement . Because of this somehow more complex scenario, and the tendency for the parasites to cause a wider variety of pathologic lesions, the various parasitic etiologies of ocular diseases will be addressed individually, including epidemiology, pathology, diagnosis, and treatment . Acanthamoeba spp . Is ubiquitous free - living protozoa that have been isolated from several habitats, including soil, bottled water, eyewash stations, and air . There are two stages in the life cycle of this environmental ameba: the motile trophozoite (840 m) and the dormant cyst (829 m). By encysting, acanthamoeba spp . Can evade extreme environmental conditions such as hyperosmolarity, glucose starvation, desiccation, extreme temperatures, and extreme ph . The leading risk factors for acanthamoeba keratitis are contact - lens wear and corneal trauma [5, 6]. Although> 80% of the cases of acanthamoeba keratitis occur in contact lens, in other countries such as india, keratitis that is not related to contact lenses commonly occurs after corneal trauma or exposure to contaminated water . One clinical manifestation of acanthamoeba keratitis is radial neuritis and severe pain that is not commensurate with the extent of tissue damage . It typically presents as a unilateral central or paracentral corneal infiltrate, often with a ring - shaped peripheral infiltrate . Other characteristic symptoms (which appear in the early phases of infection) include eyelid ptosis, conjunctival hyperemia, epithelial ulcers, and lack of discharge . These symptoms are often followed by the appearance of a ring - like stromal infiltrate in the later stages of disease . Acanthamoeba keratitis can progress to scleritis, and, in severe cases, uncontrolled infections require the removal of the affected eye . Interestingly, the pathogenic cascade of acanthamoeba keratitis has interesting parallels with the amebic colitis caused by e. histolytica . A provisional diagnosis of ak can be made using the clinical features and confocal microscopy although a definitive diagnosis requires culture, histology, or identification of acanthamoeba deoxyribonucleic acid by polymerase chain reaction . Trophozoites and cysts can be identified in giemsa or periodic - acid - schiff - stained smears from scrapings or corneal biopsy specimens . Long - term topical application of agents such as propamidine, miconazole, and neomycin has been successful in only few instances . Once introduced, the trypomastigotes circulate throughout the body with a preference for invading muscle cells, neural tissue, and the reticuloendothelial system . If the initial bite is near the orbit, the patient may experience significant palpebral and periorbital oedema (romana's sign). The edema is usually painless and is frequently followed by constitutional symptoms of fever, malaise, and anorexia . The diagnosis of acute chagas' disease is made by the detection of trypomastigotes in the bloodstream by direct examination of uncoagulated blood or buffy coat preparation . Direct culturing of blood on novy, macneal, nicolle's medium (nnn) or other suitable media may result in positive cultures in 7to 10days . Serologic testing is of little value in the diagnosis of acute chagas' disease as antibodies do not usually appear for 2to 40days following the onset of symptoms . Additionally, serologic studies may falsely detect the cross - reactivity of antibodies to nonpathogenic trypanosoma rangeli . Therapy of chagas' disease with antitrypanosome therapy is most successful in the acute stage . Therapy is usually extended for a period of months, and parasitologic cure rates are somewhat disappointing . The protozoan disease giardiasis can cause ocular complications, including salt and pepper retinal changes . One study showed that asymptomatic, nonprogressive retinal lesions are particularly common in younger children with giardiasis . This risk does not seem to be related to the severity of the infection, its duration, or the use of metronidazole but may reflect a genetic predisposition . There are numerous species within the genus, and disease manifestation is, in part, species specific . Once injected into humans during the sandfly blood meal, the promastigote develops into an amastigote after being engulfed by tissue macrophages . Within these cells, visceral leishmaniasis, or that which represents systemic disease, is known as kala - azar . The ocular manifestations of kala - azar are relatively uncommon and include chorioretinitis, central retinal vein thrombosis, iritis, papillitis, and keratitis . Glaucoma has been reported to develop after the successful treatment of kala - azar . Ocular findings in cutaneous leishmaniasis represent a local phenomenon resulting from the initial site of infection near the eye with occasional spread to the lacrimal duct . If the initial bite occurs on the conjunctival mucosa, the disease is termed mucocutaneous leishmaniasis . The diagnosis of leishmaniasis is made by direct demonstration of organisms on tissue smears or biopsy . Amastigotes are usually demonstrated fairly easily in the case of cutaneous or mucocutaneous ocular disease . However, amastigotes have not been directly identified in cases of ocular disease associated with kala - azar . When present, leishmania spp . May be cultured on novy, macneal, nicolle's medium (n.n.n .) As well as schneider's drosophila medium supplemented with 30% fetal bovine serum . While available, serologic testing is not particularly useful for diagnosing cutaneous and mucocutaneous disease due to cross - reactivity with t.cruzi and mycobacterium leprosum . Treatment of choice is pentavalent antimony, sodium stibogluconate 1520 mg per kg per day i m or iv for 1520 days . A second or even a third treatment course with pentavalent antimonal caused by the plasmodium species and transmitted via the bite of the female anopheles mosquito, this sometimes fatal infectious disease has characteristic findings in the eye . Signs of falciparum malaria in the eye include retinal whitening, retinal haemorrhage, papilloedema, and cotton wool spots [15, 16]. Much research done in endemic areas has shown a correlation between papilloedema or extramacular retinal oedema (retinopathy) and poor outcome in children with cerebral malaria . Studies show that retinopathy was associated with subsequent death, and the increasing severity of retinal signs was related to increasing risk of fatal outcome . Other studies have shown that retinal changes related to microvascular obstruction were common in adults with severe falciparum malaria and correlated with disease, severity and comma [18, 19]. It is important to emphasise that whilst these signs give a pointer to the severity of disease they do not alter the drug management of malaria . The outcome in terms of vision in patients with ophthalmological findings and severe malaria is usually good . Insights from retinal investigations have furthered the understanding of cerebral malaria [21, 22]. The photobiological effects of quinacrine and chloroquine are similar in model systems; thus, development of a bull's - eye maculopathy with quinacrine ingestion is an unsurprising potential side effect . The definitive diagnosis of malaria is made by microscopic identification of the parasite in the blood smear . A thin blood film should be examined for at least 15 minutes, whereas a 5-minute search of a thick film should reveal parasites if present . The thick film is the most efficient method of detecting malarial parasites, but interpretation requires an experienced worker . Antimalarial drugs may be classified as (1) suppressive, by acting upon asexual blood cell stages and preventing the development of clinical symptoms; (2) therapeutic, by also acting on asexual forms to treat the acute attack; (3) radical cure, for destruction of the ee forms; (4) gametocytocidal, for destroying gametes; (5) sporoniticidal, for drugs that render gametocytes noninfective in the mosquito . Two genera appear to be important in the pathogenesis of ocular disease: encephalitozoon and nosema . It should be noted that knowledge of microsporidiosis seems to be rapidly expanding, given its important role as an opportunistic infection in patients with aids . The life cycle is somewhat complex, involving three general stages: infection, merogony, and sporogony . Ocular infection is presumed to occur either by direct inoculation into eye structures or by dissemination systemically, with the latter proposed to be the pathogenesis in patients with aids . Ocular findings are generally limited to the conjunctiva and cornea . With respect to diagnosis, spores have been demonstrated in most cases in which corneal scrapings or biopsy specimens are examined by light or electron microscopy . Current recommendations for treatment include the use of albendazole, which has shown some promise in the treatment of corneal disease . Rhinosporidiosis, caused by rhinosporidium seeberi, is a mucocutaneous disease that involves the palpebral conjunctiva in ~15% of all cases of rhinosporidiosis . Reproduction of r.seeberi in tissue produces polypoid or papillary growths that arise from mucous epithelium . Recent investigations of rna genes from this microorganism disclose that it may be more closely related to fish parasites than to fungi, and it is, therefore, included in protozoan diseases of the eye . The etiologic agent, rhinosporidium seeberi, has never been successfully propagated in vitro . At present, the treatment for rhinosporidiosis is the surgical excision . Some authors proposed a medical therapy with dapsone, but the results are not convincing . Antimicrobial therapy is ineffective, and the disease may recur after months or years . The majority of infections are asymptomatic and the prevalence of antitoxoplasma igg (indicating past infection) ranges from 15 and 20% in northern europe to 80% or more in parts of the developing world . Some patients may present with a glandular fever - like systemic febrile illness with adenopathy . Most cases of adult infection will not present with eye signs, those that do usually present with a focal necrotising retinitis occasionally associated with vascular occlusion . Toxoplasmosis in immunosuppressed patients, for example, with aids can present with multiple, widespread lesions of differing chronicity and look different from classical toxoplasmosis . Vitritis is a common feature of toxoplasmosis which often leads to symptomatic haze and floaters that lasts for months after the resolution of the acute attack . Lesions are usually self - limiting, but where they threaten sight around the macula or optic nerve treatment with a combination of corticosteroids, pyrimethamine, and sulfadiazine is usually advocated . However, therapeutic trials suggest that there is little evidence that drug therapy alters the natural history of the disease . Relapses are relatively common, occurring in around 80% of patients followed up for more than 5 years . The most serious consequences of toxoplasmosis are seen when acquisition occurs in pregnancy leading to congenital infection of the newborn . Involvement of the macula is common in the developing foetus and has devastating effects on central vision . Serologic tests are very important in the diagnosis of toxoplasmosis . Because of the common occurrence of antibodies to the parasite in the general population, diagnosis by serologic means requires the demonstration of a significant increase in antibody titers . Drug treatment for ocular and cerebral toxoplasmosis is the same and lesions will continue to grow without therapy . Azithromycin has been shown to be effective in reducing the number of attacks in brazil (see tables 1 and 2). Ocular angiostrongyliasis, caused by angiostrongylus cantonensis, is a nonfatal disease; however, it can cause permanent damage to an affected eye . It was first reported in thailand in 1962 and since then has rarely presented in tropical countries . According to the a. cantonensis life cycle, the human is an accidental host . Only a small number of worms remigrate to pulmonary arteries or move randomly to other tissues such as cranial nerves or orbits . Although blood eosinophilia is demonstrated in most cases of eosinophilic meningitis, it has not been noticed in ocular angiostrongyliasis without eosinophilic meningitis . Hence, it may indicate that ocular angiostrongyliasis occurs because a worm moves randomly from the bloodstream to an eye without invasion of the brain or meninges . In cases of ocular involvement ocular symptoms although a wide range of initial visual acuity was reported, from finger count to 6/6, five cases had visual acuity less than 2/60 . The duration of visual impairment varied from 4 days to 8 weeks, mostly 2 - 3 weeks . Additionally, indirect ophthalmoscopy should be recommended in any individual presenting with a history of eating raw pila spp . No dominant affected eye has been reported because of a nonspecific pattern of parasite movement . Any types of laser, surgical removal, and corticosteroid treatment did not improve visual acuity . Alteration of the retinal pigment epithelium or retinal inflammation caused directly by parasites was the main reasons for poor vision at presentation . Furthermore, it produced permanent damage to an affected eye and gave a poor outcome . Although corticosteroids and albendazole have been reported to be effective in ocular cysticercosis and neurocysticercosis, there are no specific anthelmintic therapies in ocular angiostrongyliasis even after a definite diagnosis has been made . Human ocular infestation by live filarial worm is a rare occurrence and has been reported mostly from south - east asia . Inflammation of the retinal pigment epithelium and retinal vasculitis decreased vision, and panuveitis with secondary glaucoma can occur . Indirect ophthalmoscopy showed vitritis with plenty of vitreous membranes, and subretinal yellow lesions in the peripheral retina along with retinal pigment epithelial tracts . Baylisascaris procyonis is the common intestinal raccoon roundworm in north america and is found in 82% of raccoons in illinois . It was identified in seven childhood cases manifesting as diffuse unilateral subacute neuroretinitis and choroidal infiltrates in association with neurologic disease . Differences in inoculum level are likely responsible for isolated ocular larva migrans versus neural larva migrans in humans . Indirect immunofluorescence assays on serum, and cerebrospinal fluid is usually positive or serially positive and increasing . Dirofilarial zoonotic infections are caused by mosquito vectors that carry the parasites from their animal hosts to people . Although these infections remain rare, they are increasing in incidence and human dirofilariasis may be considered an emergent zoonosis . As the worm matures, it elicits a host inflammatory response that ultimately produces the clinical presentation of a subcutaneous nodule . Subcutaneous dirofilariasis appears as a small subcutaneous nodule that gradually grows over periods of weeks or months . When the location is ocular, the worms are situated in the conjunctiva and can be extracted by incision . The diagnosis of dirofilariasis is established histopathologically . Both the gross and microscopic features of d. tenuis have been well described . Once diagnosed, the recommended treatment is complete removal of the nematode . If the nematode is not removed, it eventually degenerates, and the mature granulomatous response results in either calcification or abscess formation with subsequent purulent expulsion of the parasite . Infection is acquired by humans through the bite of the tabinid flies of the genus chrysops . When humans are bitten, larvae pass from the fly to the human, where they develop over 1year into mature adult worms . Ocular disease may be due to both the presence of microfilaria and the presence of the adult worm . The diagnosis of loiasis is generally made by the detection of circulating microfilariae . In cases of conjunctival involvement, therapy of loiasis involves the manual removal of adult worms present in the conjunctiva in addition to the use of diethylcarbamazine (dec). Severe hypersensitivity responses may occur due to the killing of both microfilariae and adult worms . It appears that humans are the main reservoir of onchocerciasis, with infection occurring from the bite of an infected female blackfly, simulium spp . An estimated 37 million people in 34 countries in sub - sahara africa and south america are affected by it . After biting an infected person and ingesting microfilariae, the microfilariae mature to the larval stage as they migrate to the proboscis of the fly . There, the larvae may be injected into a human with the next bite, resulting in the formation of an adult worm capable of producing microfilariae . These microfilariae migrate throughout skin and connective tissue, where they die after several years . Adult worms may live in the subcutaneous tissue for years, with a female producing one - half to one million microfilariae yearly . The site of the adult worm is usually found over a bony prominence and may develop into a firm, nontender nodule, or onchocercoma . It is the migration of microfilariae through skin and connective tissue which is responsible for the majority of clinical findings in onchocerciasis . Ocular onchocerciasis is due to the presence and/or migration of microfilariae in and through ocular structures as well as the host's response to the migration . There are five predominant ocular findings that correlate with the location of microfilariae: punctate keratitis, sclerosing keratitis, iridocyclitis, chorioretinitis, and optic atrophy . Other findings may include distortion of the pupil, which may also be covered with exudate . Wolbachia and wolbachia - derived molecules are bacterial symbionts of o. volvulus that is implicated in the pathogenesis . Experiments using wolbachia - containing extracts of o. volvolus in a mouse model of onchocercal keratitis demonstrated that the presence of the bacteria was essential for neutrophil - mediated inflammation, opacity, and corneal haze . The diagnosis of onchocerciasis is accomplished by a combination of clinical symptoms and signs with histopathologic examination of specimens . Pcr may aid in the diagnosis of disease associated with a low burden of microfilariae . Xenodiagnosis, using laboratory - bred blackflies, may provide a clue as well . Traditional therapy has centered on the use of dec, but this is active only against microfilariae, allowing adult worms to repopulate the microfilariae in several months . Ivermectin is the treatment of choice and mass distributed by the who onchocerciasis control and the onchocerciasis elimination programme for the americas . This had led to dramatic improvements in disease control to the extent that elimination has become a realistic target . Transmission of eyeworms occurs via nonbiting diptera that feed on the ocular secretions, tears, and conjunctiva of animals . The disease, thelaziasis, is characterized by a range of subclinical to clinical signs such as epiphora, conjunctivitis, keratitis, corneal opacity, and ulcers . Asymptomatic, subclinical thelaziasis occurs mainly when only the male nematodes parasitize animals, whereas evident symptoms have been more frequently registered in the presence of gravid females . The lateral serration of the thelazia cuticle causes mechanical damage to the conjunctival and corneal epithelium . T. callipaeda nematodes have a serrated cuticle, buccal capsule, mouth opening with a hexagonal profile, and 6 festoons . Fortreatment of human cases, the removal of the worm is suggested . Topical treatment with thiabendazole hasalso larva migrans in man are a disease characterized by inflammatory reaction around or in the wake of migrating larvae, most commonly larvae of nematode parasites of other animals . For some of the larva migrans producing larvae, man is merely an accidental but more or less normal intermediate or paratenic host . Toxocariasis is an important cause of unilateral visual loss and leukocoria in infants, and as a differential diagnosis of retinoblastoma . Visceral larva migrans are best known in the form produced by the larvae of toxocara canis, these having been identified in autopsy specimens of lungs, liver, brain, and in several enucleated eyes . Human infection by a spiruroid form of nematode gnathostoma spinigerum has been reported sporadically from thailand, the philippines, china, japan, and india . Intraocular parasites occur so rarely that they are considered as ophthalmological curiosities, nevertheless, it can cause intraocular hemorrhage, uveitis, and loss of vision within 2 days . Following surgical removal, treatment is with albendazole and topical corticosteroids . Trichinosis is a parasitic disease which probably presents itself for diagnosis not infrequently . Because of its varied symptomatology trichinosis is, unless by chance, almost as frequently undiagnosed ocular trichinosis can manifest itself as oedema of the face especially around the eyes, conjunctivitis, and exophthalmoses . Diagnosis is only confirmed by finding the worm in a section of the excised muscle (see tables 3 and 4). This helminthic infection caused by the larval cysts of the pork tapeworm (taenia solium). Ocular involvement is well recognised and includes orbital, intraocular, subretinal, and optic nerve lesions [5457]. Cysticercosis can be evident as a free - floating cyst with amoeboid movements within the vitreous or anterior chamber of the eye . Gaze palsies may also occur secondary to intramuscular cysts or cranial nerve lesions from intracerebral cysts . Diagnosis depends on imaging with ultrasound, mri, and ct scanning all being useful, depending on the location of the cysts [58, 59]. Serology can be useful but in cases of isolated cysts may be negative . Treatment is largely with the antihelminthic albendazole . Antihelminthic therapy may lead to an increased inflammatory reaction around the lesions, and for this reason corticosteroids are often used when treating neurological or ocular disease . Spontaneous extrusion of cysts from the orbit may occur, and surgery may be required for isolated ocular lesions when they are growing and causing visual loss . Fasciola hepatica is a zoonotic helminth that is prevalent in most sheep - raising countries . In 1989, an outbreak of human infestation of more than 10 000 cases living in guilan province, iran was reported . The biliary duct of the liver is the main site of establishment of the parasite . However, immature flukes may deviate during migration, entering other organs, and causing an ectopic infestation . In humans, ectopic locations in the orbit [62, 63] identification of the route of entry of the parasite larva into the anterior chamber of the eye is difficult . One possible route can be via the central retinal artery into the vitreous, causing vasculitis and endophthalmitis . Severe intraocular reaction, haemorrhage, diffuse vasculitis, and retinal ischaemia of the patient may be caused as a result of the presence or irritation of the parasite . Early vitrectomy and removal of the parasite resulted in a rapid response, with reasonable final visual acuity . Hydatid cysts are most commonly seen in the liver (6070%) and lungs (20%). Hydatid disease involving the orbit represents <1% of all cases of hydatid disease and requires surgical treatment . Laboratory and immunologic tests are generally unhelpful . From the literature and our own observations, orbital hydatid cysts usually appear as a well - defined, thin - walled, oval shape lesions with fine peripheral rim enhancement of their fibrous capsule after contrast medium administration . Various theories have been postulated as to the different routs by which the schistosoma ova or even the adult worms can reach the systemic circulation and then after lodged in ectopic sites such as the eyes . Cercariae (the infective stage) develop to maturity and lay their eggs in the veins directly under the skin or the mucous membrane through which they have penetrated if the part is richly vascularised . The presence of schistosomal eggs in the eye can produce granuloma formation and inflammatory sequelae . Considering how common the infection is in endemic areas, involvement of the eye is incredibly rare . Effective treatment, using the drug praziquantel, has been available for 25 years, but the growth of human populations in high - risk areas, as well as the high probability of rapid re - infection after treatment, has thwarted efforts to control the number of human infections worldwide (see tables 5 and 6). Genera important to human myiasis include dermatobia, gasterophilus, oestra, cordylobia, chrysomia, wohlfahrtia, cochliomyia, and hypoderma . Ophthalmomyiasis may be categorized into three categories: ophthalmomyiasis externa, ophthalmomyiasis interna, and orbital myiasis . Ophthalmomyiasis externa is usually seen in areas of shepherding and is typically due to larvae of the sheep nasal botfly, oestra ovis . A crawling or wriggling sensation accompanied by swelling and cellulitis orbital myiasis may be due to a number of fly species and is generally seen in patients who are unable to care for themselves . Diagnosis of ophthalmomyiasis is made by demonstration of maggots, and histologic examination may show granuloma formation . Capitis, the human head louse; phthirus pubis, the crab louse . Depending on the species, eggs, or nits, are laid and glued to body hairs or clothing fibers . Following this, nymphs emerge to feed on the host, giving rise to symptoms of pruritis . Of the species in addition to pruritis, small erythematous papules with evidence of excoriation may be present . Involvement of the eyelash may cause crusting of the lid margins . In this case, diagnosis is relatively simple as nits are easily seen at the base of the eyelash . Eyelid disease is treated with a thick layer of petrolatum twice a day for 8days, or the application of 1% yellow oxide of mercury four times a day for 2 weeks . There are a number of different species of ticks which may cause disease in humans and animals . Ticks exist in three life stages larva, nymph, and adult all of which requires blood meals . Most tick bites are uncomplicated, and prompt removal of the tick is all that is necessary . In one such case, the nymph was associated with a stinging sensation . Following the removal of the tick, a firm nodule, representing a tick bite granuloma, may remain for several weeks . The majority of the clinically important species of parasites involved in eye infections are reviewed in this paper . Emphases have been placed on literatures published within the past decade, but prior noteworthy reviews and case reports are included . We searched the medline database via pubmed and identified articles by cross - referencing the terms ocular, eye, ophthalmic, retinitis, endophthalmitis, conjunctivitis, and uveitis to specific infectious diseases in adults . We searched the cochrane database for systematic reviews on the treatment of specific parasitic ocular infections . Additionally, we reviewed texts for completeness and to obtain other references of eye complications of systemic infections (see tables 7 and 8).
The majority of known genetic causes of craniosynostosis are mutations in the genes encoding fibroblast growth factor receptor types 1 - 3 (fgfr 1, 2 and 3); other significant genes are twist1 and efnb1 . A major breakthrough in understanding the genetic background of craniosynostosis has been the identification of genetic defects in several syndromes, including the most common crouzon syndrome and apert syndrome . In general, more than 40 genes and several variants were reported in the literature as genetic risk factors for craniosynostosis and only 60% heritability was explained . The presence of mutations in the group of genes coding for the fgfr in patients with apert and crouzon syndromes is now clearly established . These genes code for receptors on the cell surface, which mediate the effects of fibroblast growth factors (fgf). The effects of fgfs are not fully understood, but they are already clearly implicated in important cellular processes such as cell growth, differentiation, migration and survival . Although 4 different genes are located in different chromosomes, the receptor proteins they encode for being very similar structurally . A number of craniosynostotic disorders have recently been ascribed to mutations in genes coding for fgfr 1, 2 and 3 . The common features of these fgfr associated conditions are the unilateral or bilateral premature ossification of the coronal suture . The present finding points out the importance, from both diagnostic and prognostic points of view, of early fgfr mutational screening in craniosynostotic conditions, even in forms that apparently do not involve closure of the coronal suture at birth . The aim is to identify association of mutation of fgfr1, fgfr2 genes with syndromic as well as non - syndromic craniosynostosis in indian population . The aim is to identify association of mutation of fgfr1, fgfr2 genes with syndromic as well as non - syndromic craniosynostosis in indian population . The aim is to identify association of mutation of fgfr1, fgfr2 genes with syndromic as well as non - syndromic craniosynostosis in indian population . Retrospective analysis of records of patients registered in craniosynostosis clinic from january 2008 to december 2012 was done . Ethical clearance from the institute's ethical committee was taken . Diagnosed cases of syndromic and non - syndromic craniosynostosis patients between 6 months and 12 years of age either pre - operative or postoperative were included in the study . Patients with primary microcephaly (secondary craniosynostosis), postural plagiocephaly, incomplete data and lost to follow - up were excluded from the study . Diagnostic investigations include clinical examination and plain x - ray skull (anteroposterior, lateral and towne's view) and non - contrast computed tomography with 3d reconstruction if required . Out of 63 registered cases, 41 satisfying the inclusion were taken for the study . Blood sample (3 ml) was taken from both the parents along with the child in ethylene - diamine - tetra - acetic acid vial . For control 50 healthy children of comparable age group, belonging to the same geographical region genomic deoxyribonucleic acid (dna) was extracted from peripheral blood lymphocytes by phenol chloroform extraction method . Primers to diagnose common fgfr1 and fgfr2 mutations in this study are listed in table 1 . Custom - synthesized primers for fgfr1 and fgfr2 gene were designed (sigma aldrich chemicals pvt . Ltd ., polymerase chain reaction (pcr) for each sample was performed in 0.2 ml, thin - walled tubes using 20 ng of dna, 2 - 5 pmol of each primer, 200 mm dinucleotide triphosphates, 10 pcr buffer, 1.5 mm mgcl2 and 0.5 units of dynazyme ii dna polymerase (thermo scientific). The pcr reaction was carried out in a t-100 dna engine (bio - rad, hercules, ca, usa) thermal cyclers under the following conditions: 95c for 3 min, 35 cycles at 95c for 30 s, annealing temperature as in table 1 for 30 s and 72c for 1 min / kb and a final extension at 72c for 7 min . Amplicons size were verified by gel electrophoresis by running the pcr product on 2% agarose gel with the 100 bp maker (ladder). After successful amplification, pcr products were digested as per the manufacturer's instructions with the respective restriction endonucleases mentioned in table 2 and analyzed on an ethidium bromide - stained 3.0% agarose gel with 50 bp ladder . Primer sequences used for present study fgfr1 and fgfr2 amplicon size (bp) after pcr amplification and restriction digestion there were 33 (80.4%) non - syndromic while 8 (19.5%) syndromic cases . Out of these 8 syndromic cases, 4 were apert, 3 were crouzon and 1 pfeiffer . Phenotypically the most common non - syndromic craniosynostosis was scaphocephaly (19, 57.7%) followed by plagiocephaly in 14 (42.3%). Fgfr1 mutation (pro252arg) was seen in 1 (2.4%) case of non - syndromic craniosynostosis while no association of mutation with either fgfr1 or fgfr2 mutation was noted in syndromic cases . The only mutation fgfr1 (pro252arg) seen, was in a boy with non - syndromic unicoronal craniosynostosis . The mutation was also present in her mother who had mild facial asymmetry, but do not have craniosynostosis . Figure 1 showing the pcr product amplification of fgfr1 on 2% agarose gel, after successful amplification the product was digested . The homozygous normal allele (cc) produced 4 fragments of 109 bp, 69 bp, 27 bp, 11 bp size . The heterozygous (gc) produced 5 fragments of 109 bp, 136 bp, 69 bp, 27 bp, 11 bp size, whereas the homozygous mutant (gg) produced 3 fragments of 136 bp, 69 and 11 bp . However due to 69 bp, 27 bp, 11 bp are too small to be visualized so the normal, heterozygous and mutant allele were defined on the basis of bigger fragments, i.e. 109 and 136 bp . Lane 1 contains the pcr product of size 216 bp, lane 2 contains the heterozygous condition (gg) size 109 and 136 bp for for craniosynostosis children and lane 4, 5 shows digested pcr product of size 109 bp for non - craniosynostosis children [figure 2]. 2% agarose gel showing polymerase chain reaction (pcr) product of fibroblast growth factor receptor 1 amplification (216 bp) (1) molecular maker (100 bp); (2 - 7) pcr prdocut of craniosynostosis and and non - craniosynostosis children restriction fragment length polymorphism of polymerase chain reaction (pcr) product of fibroblast growth factor receptor 1 on 3% agarose gel from craniosynostosis and non - craniosynostosis children's which were analysis for mnli digestion . (1) molecular marker (50 bp); (2) pcr product for craniosynostosis children's; (3) completely digested pcr product for craniosynostosis children's (gc) (136 bp, 109 bp); (4 and 5) completely digested pcr product for non - craniosynostosis children's (cc) (109 bp) children suffering from genetic disorders are not only social liability, but also an extra burden on the economy of developing countries such as india, pakistan and sri lanka . Craniosynostosis is one of the major genetic disorders affecting the central nervous system in children with reported incidence of 1/2,200 live births . Since then multiple theories have been proposed to explain the pathogenesis, with recent studies focusing on genetic regulation . Still the etiology of the disease is largely unknown however the condition is related to abnormalities in the base of the skull and is frequently seen in association with osseous abnormalities of the face . Universally accepted hypothesis is an abnormal premature fusion of the cranial suture mainly because of an imbalance between proliferation and differentiation of cell . Craniosynostosis can either be non - syndromic or syndromic with the former more common than the latter . Various authors of the western world have reported the role of fgfr1 and 2 in craniosynostosis . Our study is truly based on indian population suffering from craniosynostosis . In english literature, multiple reports are available regarding the role of fgfr1 and fgfr2 genes in craniosynostosis, but similar data from the indian subcontinent is still lacking . To the best of our knowledge, this is the pilot study from asian subcontinent addressing the association of the fgfr1 and fgfr2 mutation with craniosynostosis . In the present study, we have screened the most common associated mutation of fgfr1 pro252arg and s252w in fgfr2, pro253arg in fgfr2, q289p, arg342cys in indian population . Pedigrees of 41 children suffering from both syndromic as well as non - syndromic craniosynostosis (apert syndrome, crouzon and pfeiffer syndromes) were analyzed . We did not find any association with fgfr2 mutation in families of children suffering from craniosynostosis . Only mutation (fgfr1 [pro252arg]) seen, was in a boy with non - syndromic unicoronal craniosynostosis . The mutation was also present in his mother who had mild facial asymmetry but do not have craniosynostosis . Age of the child was 32 months and on examination there was flattening of the frontal bone on the right side, with a prominent frontal eminence on the left side resulting in anterior plagiocephaly . There was no exorbitism, midface hypoplasia, oral palatal defect, or ear abnormality either in mother or son . The child does nt show any peculiar features to differentiate him from others except the manifestation of underlying primary pathology . Most patients with craniosynostosis do not, however, have obvious syndromic features, making an accurate diagnosis and genetic counseling more difficult, particularly because non - syndromic craniosynostosis is likely to be etiologically heterogeneous . Up to 30% of such patients with coronal craniosynostosis have a specific mutation in fgfr3 (pro250arg) that is more reliably identified by genetic testing than by clinical features . Mutations of fgfr2 are much rarer in non - syndromic patients, but a small number of fgfr2 mutations have been identified in individuals with mild, atypical or more variable phenotypes . Our study provides evidence that the mutation is not associated with craniosynostosis in indian children, although it has been shown to be associated with children in australia and other western countries . Because india is known for its diversity and complexity of genome therefore, it is important to perform replicate studies of patients from diverse ethnic origins (different states of india) before either designating or excluding this mutation as a risk for craniosynostosis . This study will help in better understanding of the existing genetic mutations in indian children suffering from this grave disease thereby providing an opportunity to the treating pediatric surgeons to reduce the agony and suffering of the children . The present study tries to establish a novel bimolecular marker for consideration and determination of craniosynostosis patients in india . Our study has its own limitation such as small sample size for arriving at any definitive conclusion . In a recent study, wilkie et al . Has shown the prevalence and complications of a single gene and chromosomal disorders in craniosynostosis . They concluded that cytogenetic and molecular genetic testing, as a minimum for mutations in fgfr3 (p250r) and fgfr2 exons iiia and iiic, should be an integral part of management in children with bicoronal, unicoronal or multisuture synostosis . Research aimed at identifying new genetic mutation in craniosynostosis requires careful choice of the patients, as many with chromosomal abnormalities or other syndromes may have secondary causes . Other publications on craniosynostosis involving european ancestry (nhw) populations were important from the perspective of population specific understanding of the genetic causes . Justice et al . In their study of 130 non - syndromic cases showed the susceptibility loci for non - syndromic sagittal craniosynostosis near bmp2 and within bbs9 and was associated with familial (case - parent trios of european ancestry) craniosynostosis . It also represented the first major step toward deciphering the genetic etiology of non - syndromic sagittal craniosynostosis (sins). Reported that alx4 variants may have an impact on the genetic etiology of non - syndromic craniosynostosis . Seto et al ., in a series of 164 cases have shown that genetic testing of patients with isolated sagittal or coronal synostosis should include twist1 mutational analysis . Study of asian continent through korea by yu et al . Showed the genotypic and phenotypic analyses of korean patients with syndromic craniosynostosis . Our study provides the strongest evidence that association of mutation of fgfr1, fgfr2 with syndromic as well as non - syndromic craniosynostosis does not exist in indian population as seen in western population . Our study will provide the necessary platform for further research to better understand the genomics of craniosynostosis in indian population.
Research in the last decades has shown that the more involved patients have better health outcomes, due to their increased treatment adherence and awareness to preventive tests . In addition, the availability of information has changed patients' expectations regarding the extent to which they should be involved in decisions on their own care [1, 2]. Medicine is becoming participatory: patients are increasingly engaged and active participators in personal choices about illness and well - being . One of the major sources of health information in recent years is the internet . In the us, 75% of internet users (about 60% of the total population) nevertheless, there is a gap between the number of patients actually searching for information and those reporting what they found to their physicians . Therefore some patients are exposed to information that is not reliable, and of which their physicians are not aware nor able to provide their reaction, or opinion, [6, 7]. To date, only few studies have assessed the issue of internet usage by patients; atreja et al . Reported that patients usually look for information before and after medical visits and use the internet to understand medical terms . In another study, reported that the majority of ms participants searched for information on the internet prior to the first visit to an ms clinic . Internet activity was correlated with income but not with education, marital status, health status, or gender . Although information found on the internet did not replace the information obtained from the physician, two - thirds of the patients were reluctant to discuss internet information with their physician . The authors suggested that patients were concerned that online searches might be perceived as a lack of confidence in the physician skills . The fact that patients are unlikely to discuss search results with physicians may have implications for patient adherence to treatment . The quality and information content of websites available for the ms patient was found to be variable, with a few sites offering nearly all of the information needs of people with ms . A few websites, as the one from the ms centers of excellence, go beyond information and support self - monitoring of the disease . Since ms patients experience a vast array of symptoms that may exacerbate or decrease over time and may suffer also from memory impairment, detailed registration of the symptoms can improve disease management, by allowing more accurate reports to the physician . A similar system was developed by lowe - strong and mccullagh who built a computerized visual interface for self - recording of pain associated with ms . Ms patients expressed interest in a web - based portal that would include self - monitoring of ms symptoms, prescriptions orders, laboratory results retrieval, online patient education, updates on ms research, and, most importantly, timely communication with the medical team [8, 13]. Ms prevalence in israel is in the range of the medium to high zone, with over 3000 patients currently registered in the israeli national ms register . Although the internet is frequently used in israel by an estimated 56% to 70% of the population [15, 16], to the best of our knowledge there are no reports on the use of the internet for seeking health information by patients, and specifically by ms patients . The objectives of this paper were (1) to assess the percentage of internet users and the percentage of seekers of information on ms among the patients of our clinic, (2) to characterize the topics on ms most searched in the internet and the outcomes of these searches, (3) to assess the patients' attitudes towards internet information, and (4) to assess the impact of disease duration and disability on information seeking . Ms patients who visited the ms clinic at carmel medical center, a major referral ms center in northern israel, during the spring of 2009, who were 18 years and older and able to communicate in hebrew, were invited to participate in the study . Patients willing to participate completed questionnaires about internet reliability and accessibility, their browsing habits, demographic data, and their opinion about the medical team's attitude to the information they collected through the internet . The study received ethics approval from carmel medical center's helsinki committee, and all participants received explanations on the study objectives and signed informed consents . Comparisons between groups of patients were done by using one way anova followed by independent t - test for the continuous variables, and by kruskal - wallis followed by mann whitney for the ordinal variables . Chi square test or chi square exact test were used as appropriate for the categorical variables . A total of 103 patients agreed to participate and 96 (93%) completed the questionnaire . As in other studies based on self - filled questionnaires, response rate was not the same for all questions, and therefore percentage of respondents was calculated separately for each question . The average age of patients was 43.2 years and female / male ratio was 2.4, similar to the ratio reported in other studies . Sixty - two percent of the participants (56 out of 90) had more than 12 years of education which is lower than that reported in studies of ms patients in other countries [13, 1821]. Mean disease duration since diagnosis was 7.7 years, and mean edss (expanded disability status scale) was 2.6, indicating a mild disability level for this patient cohort . Although in this paper only patients fluent in hebrew were recruited, participants included a variety of ethnicity groups and countries of origin, characteristic of the israeli population . Sixty - one patients (63%) used the internet and searched for topics related to ms (ms internet users- msiu). Eighteen patients (19%) used the internet for general issues not related to ms (general internet users- giu) and 17 patients (18%) did not use the internet (noninternet users niu). Comparison of demographic and clinical data between the 3 groups of patients can be seen in table 1 . The main differences between niu and msiu were the older age and longer disease duration of the niu group . Among niu, the principal reasons stated for not using the internet to search about ms were lack of computer operating skills (6 out of 17 patients, 35%), lack of knowledge on computer search tools (4 out of 17 patients, 23%), or lack of access to a computer (4 out of 17 patients, 23%). Among giu the principal reasons were lack of interest in information about ms (8 out of 18 patients, 44%) and lack of knowledge on search tools (4 out of 18 patients, 22%). The physician and the nurse were the principal source of information on ms for patients in all 3 groups: 94% of niu (15 out of 16 respondents), 87% of giu (13 out of 15 respondents) and 94% of msiu (47 out of 50 respondents). Msiu also searched more significantly for information on newspapers than giu (3 out of 15 respondents, 20%; compared to 27 out of 49 respondents, 55%; p = .02) and in the television (4 out of 14 respondents, 29%; compared to 22 out of 47 respondents, 47%; p = .03). The most common source of information on the disease for niu was leaflets (7 out of 11 respondents, 64%). Over 80% of participants in both groups browsed at least two times a week and browsed in hebrew sites . The most frequent uses of the internet were for email (16 out of 18 giu patients, 89%, and 54 out of 59 msiu patients, 91%), work - related issues and tourism, and entertainment . Regarding the usage of online health services (not related to ms), although the majority of patients in both groups reported the usage of the internet for setting medical appointments and retrieving lab results, the groups differed in their use of search services: msiu used the internet more frequently to search for physicians (32 of 47 msiu patients, 68%, compared to 4 of 12 giu patients, 33%, p = .04) and to search for medicines, diseases, and medical tests (47 of 52 msiu patients, 90%, compared to 6 of 13 giu patients, 46%, p = .001). There was high variability in the percentage of msiu patients interested in different information topics (figure 1). Over 90% of the patients searched for information in order to understand the disease and to find a treatment . The lower rates were for interactive topics such as support groups / contact with other patients and interaction with specialists . The principal outcomes following searches in the internet were patient confidence in the treatment received and better coping with the disease (figure 2). Similarly to what was reported in other studies [6, 7, 9], only one - third of the patients discussed the information they gathered on the internet with their physician . The websites most visited for searches related to ms were patients' associations sites (73%36 of the 49 respondents) and academic sites (69%36 of the 52 respondents). Commercial medical sites were visited by 26 out of 51 respondents (51%), pharmaceutical companies' sites were visited by 20 out of 43 respondents (46%), health maintenance organizations (hmos) sites were visited by 17 out of 47 respondents (36%), and hospital sites were visited by 11 out of 43 respondents (26%). Giu and msiu attitudes towards information, the internet, and the medical team's attitude to information gathered on the internet were compared . Participants were asked on a 4 point scale if they do not agree at all (1) or completely agree (4) to each one of the attitudes' related statements . Responses 1 and 2 were grouped into do not agree and responses 3 and 4 were grouped into the results indicate that patients rely on online information and are satisfied with the information available to them . Most patients stated that information about ms contributed to their ability to cope with the disease, especially among msiu, and only about one third of patients in both groups stated that information about the disease frightens them . Although participants did not feel encouraged by the medical team to search for information, as reported also in other studies, about half felt the medical team was happy to discuss with them information they found and the majority reported feeling part of the decision the making process . In order to assess if the information needs of msiu change according to disease duration, we compared disease durations for patients that searched for information on each of the topics presented in table 1 and those that did not include that topic in their searches . No statistically significant difference in disease duration was found in any of the search topics . However, two of the attitude statements studied (presented in table 2) appeared to be influenced by the disease duration of the patients: the disease duration of msius who stated that they increase their searches for information when the disease worsens was longer than that of patients that did not state so (mean 7.8 years compared to 4.3 years, p = .02), and the disease duration of msius who stated that they feel part of the treatment decision making was significantly longer than that of patients who did not state so (mean 7.2 years compared to 4.5 years; p = .04). For assessing if the information needs of patients are related to disease disability, patients were grouped according to their edss score in 3 groups: lower or equal to 3, between 3 and 5.5, and equal to or higher than 5.5 . We tested for each of the topics searched if there is a difference between the edss scores of patients who searched information on that topic versus patients that did not include the topic in their searches . Patients with edss 5.5 and higher were significantly more interested in interacting with specialists than patients with 3 <edss <5.5 (4 of the 6 respondents, 67% compared to 1 of the 10 respondents, 10%; p = .04). High edss score patients were also more interested in support groups via the internet, than patients with 3 <edss <5.5 and patients with edss 3 (4 of the 6 respondents, 67%; 2 of the 9 respondents, 22%; and 7 of the 34 respondents, 21%, resp ., p = .06). Regarding patients' attitudes to information, patients who were more disabled appeared to be more interested in reading about other patients' coping strategies with ms . All 6 patients with edss 5.5 and higher expressed interest on this topic, whereas the patients in the lower edss groups displayed a trend of decreasing interest with the lower disability levels (10 patients of 12 respondents with edss 3 <edss <5.5, 83% and 23 out of 42 respondents with edss 3, 56%, p for trend = .01; figure 3). The main findings of this study were that the majority of ms patients browsed the internet for information on ms, and their information needs were correlated to disease duration and severity . Participants considered online information as accessible and reliable, and claimed it helped them cope with ms and raised confidence in their therapy program . Most ms patients from our clinic that browse the internet search for information related to the disease (msiu group), similarly to findings from previous studies on ms [9, 23] and other chronic diseases . Nevertheless, the medical team remains their principal source of information . Among the noninternet users (nius), the reason for not using the internet was mainly lack of computer skills, similarly to findings of a previous study of cancer patients, which is in line with their older average age compared to msiu patients who searched for ms topics . Interestingly, although the giu patients had internet skills that were similar to the msiu patients, yet they did not search for information on ms, mostly due to lack of interest . Although the average age of this group was similar to that of the niu group, since their internet skills resembled that of the msiu group, their age difference may indicate an age - dependent attitude biased towards the physician - dependent paternalistic healthcare approach . A study with a larger number of giu is necessary to better understand the reasons for their internet behavior . In any case, it should be clarified that appropriate information on the disease and the treatments given by physicians remains the main goal to reach . The majority of patients considered information on ms available on the internet to be as reliable as in books and more accessible . The reliability and credibility of health information on the internet and its effect on healthcare have been frequently discussed . There is a general assumption that low - quality information on the internet may lead to potential harm, although no evidence can be found in the literature . Studies that measure the impact of information on healthcare objectively, for example, by measuring health status of informed compared to less informed patients, are difficult to perform, due to the presence of confounding factors such as participants' interest in online information and health status of participants before the intervention . Due to the limited number of participants in this study, a consequence of the interval time stipulated for recruitment, the effect of confounding factors like disease type and severity on change of health status could not be assessed and the evaluation of the contribution of online information to healthcare was based on the patients' self - impression of benefit . No harmful effects of the information on the internet were reported, rather, the information was perceived to have positive effects . The first one was the ability to cope with the disease, which has already been connected to how informed the patient is . . Reported on a study on ms - related coping styles that optimizing the information process, especially in the early phase of the disease, may induce coping styles that produce a better adaption to living with ms . Another positive effect of information seeking was confidence in the treatment received, which can improve treatment adherence . Lately, the term adherence has replaced the term compliance with respect to treatment, in order to emphasize the importance of the patients' independent role in treatment decision - making process . Nonadherence is seen as an unnecessary risk for further morbidity and mortality, as well as a waste of health care resources, and is a significant issue in ms management . Information is an important key to patient empowerment in decision making and optimization of therapy, contributing to increased adherence . This is especially true when multiple treatment options are present, as in ms . Most participants did not discuss the online information with their physicians, similar to findings of other studies on ms, rheumatologic diseases, and cancer the physician therefore may not be aware of unreliable information the patients may have been exposed to, which may have affected their coping behaviors, and accordingly, disease management . Our finding that patients with shorter disease duration tended to search more for information on the internet reinforces studies that have emphasized the importance of supplying information to recently diagnosed ms patients [28, 31]. On the other hand, patients with longer disease duration searched more for information when the disease worsens, an indication of the change of information needs over time . They also felt more part of the decision making in their treatment, which may be due to a longer interaction over the years between the patient and the medical team . Patients with higher disability expressed preference to interaction through the internet with specialists and support groups, and were more interested in reading about coping approaches of other patients with the disease . This may indicate that the internet serves a social role for these patients, whose disability limit their accessibility to social contacts, as well as to medical support systems . These results emphasize, as suggested also in previous studies [32, 33], that information should be tailored to suit patients according to their needs, and that the accessibility to online information is of exceptional importance to the disabled . The development of quality websites to a patient's population requires the knowledge of the specific conditions related to their disease . Furthermore, data on browsing habits including frequency, languages, and use of online services can allow development of sites adequate to these habits . Another issue to be taken into account is the ms patient's limitations in accessing the internet, due to disease - related impairments such as visual disturbances, fatigue, and cognitive and memory problems in ms . Physicians then can take advantage of the availability of reliable sites by referring patients to appropriate information and discussing the information as part of the health management plan . The results presented here can contribute to the development and implementation of websites dedicated to ms patients.this paper outlines the information topics of most interest for ms patients and the association between information needs and disease stage . Ms clinics may improve medical care by providing relevant information which is patient - centered and online as possible . Additionally, ms patient societies, hmos, and pharmaceutical companies, may play an important role as information sources by maintaining informative and updated websites, catered to patient usage . The internet could also be used for patients education, for example, by providing tutorials on how to inject medications and potential adverse events, and could serve as a tool for data collection in ms research, through the use of internet - based surveys . When developing internet resources for ms, it should be taken into account that some patients do not use the internet due to lack of the necessary skills . These patients should be identified by the medical team and should either receive the necessary support to be able to use this important tool or should be provided with the relevant information through other means . The results of this study, which to the best of our knowledge is the first to present an association between ms patients' clinical and demographic characteristics and their attitudes and browsing behaviors with respect to disease - related information on the internet, indicate that internet usage is well accepted by ms patients and contributes to patients' well being . Although internet based information is not often discussed with the treating physician, extending the patient - physician interaction to the web is likely to increase patient accessibility to reliable information, and contribute to their health management . Internet usage may contribute to improved and personalized healthcare by offering information that suits the patient's specific needs and perceptions regarding the stage of his / her disease, impairment, socio economic and computer literacy level, and ethnicity viewpoints . Additional studies, with larger number of participants and objective assessment of information effect on health status, will contribute to development of useful patient - oriented sites that will aid in positive coping strategies . Tailored information will lead to more empowered patients towards the goals of modern medicine: to be both personalized and participatory.
Upon reviewing the literature, it was found that nasopharyngeal angiofibroma and meningo - ophthalmic artery (m - oa) anomaly are relatively rare phenomena . In denmark, the incidence of nasopharyngeal angiofibroma is reported to be 3.7 per million males per year . Tumor extirpation is the mainstay treatment, but the recurrence rate (20%, ranging between 5 and 50%) remains a challenge for the surgeon . In the case of m - oa anomalies, the ophthalmic artery (oa) variant may supply the orbit completely or partially, in the absence or presence of a normal oa with internal carotid artery (ica) origin, respectively . Retinal supply from the middle meningeal artery (mma) was also encountered . The following incidences for oa origin from the mma have been reported: 1.18% among 170 specimens, based on dissection studies in the usa; and 1.45% among 1652 oas, based on magnetic resonance imaging (mri) studies in japan . To the best of our knowledge, an m - oa anomaly has not been previously reported in a patient with nasopharyngeal angiofibroma . Awareness of vascular collateral routes with routine angiography can effectively contribute to strategic planning and surgical interventions for tumor extirpations . Embolization of the external carotid artery (eca) is a useful tool in the devascularization of nasopharyngeal angiofibroma and in the treatment of epistaxis . However, the surgical interventionist needs to be aware of dangerous eca ica connections to avoid neurological complications such as embolic stroke and palsies . Among the potential orbital collateral routes the m - oa is the most frequent followed by a route from the anterior branch of the mma . And a route from the anterior branch of the mma . For embolization procedures, it is imperative to identify the source of the retinal supply to avoid visual complications . This paper reports on unilateral m - oa and mma orbital collateral routes as the sole supply of the left orbit in a patient with nasopharyngeal angiofibroma . Questions pertaining to the orbital collateral routes are the following: why do they exist? A 12-year - old boy was admitted to our academic hospital and diagnosed with nasopharyngeal angiofibroma . Computed tomography (ct) scanning revealed a midline nasopharyngeal angiofibroma with prominent right - side infratemporal fossa infiltration [figure 1]. Magnetic resonance imaging (mri) confirmed exclusion of intra - orbital infiltrations [figure 2]. A lateral rhinotomy to remove the tumor was required . Access to the right common transfemoral artery was gained with a 22 g jelco, and a micro - puncture guide wire was advanced into the right common femoral artery . A 4 f pediatric sheath was advanced over the micro - puncture guide wire into the right common femoral artery . A headhunter catheter was used and 50 ml of jopamiron 300 was administered intra - arterially without any side effects . A six - vessel study, i.e. Eca, ica, and vertebral artery (va) on both sides, was selected to determine the blood flow routes of the angiofibroma for optimal tumor extirpation . Dangerous eca ica connections rendered embolization of prominent feeder arteries unsafe, but super - selective fibrin glue embolization of both sphenopalatine arteries with a micro - catheter was performed in an attempt to control epistaxis . Post - operatively, the child still suffered from epistaxis because of the extensive nasal anastomotic networks encountered . The child had a difficult recovery, but was eventually discharged and scheduled for follow - up visits . Twelve - year - old boy presented with nasopharyngeal angiofi broma diagnosed with unilateral meningo - ophthalmic artery anomaly . Computed tomography scan shows midline nasopharyngeal angiofi broma (black arrow), with prominent right - side infiltration into the infratemporal fossa (white arrow). Twelve - year - old boy presented with nasopharyngeal angiofibroma diagnosed with unilateral meningo - ophthalmic artery anomaly . The pre - operative angiographic study revealed the following prominent feeder arteries: ascending pharyngeal artery with multiple pharyngeal branches from the pharyngeal trunkus (bilateral), proximal maxillary artery (ma) with branches from the stem of the m - oa (left side) and the mma and accessory meningeal artery (ama) (right side), distal ma with sphenopalatine artery (i.e. The nasopharyngeal artery) (bilateral), and distal facial artery with nasal branches (bilateral). According to expectation, ethmoidal and nasal branches via the oa (i.e. The left oa variant and the right oa with ica origin) and nasal branches via the distal facial artery (unilateral) nourished the midline nasopharyngeal angiofibroma . For the purpose of this paper, prominent feeder arteries and two major vascular networks are given in table 1 . Tumor feeder arteries, branches, and vascular networks twelve - year - old boy presented with nasopharyngeal angiofibroma diagnosed with unilateral meningo - ophthalmic artery anomaly . Right lateral view angiograms of head show a) pre - embolization of tumor vessels and b) post - embolization of tumor vessels . Left lateral view angiograms of head show c) pre - embolization of tumor vessels and d) post - embolization of tumor vessels . Left angiograms revealed no oa with ica origin, and an m - oa anomaly that originated from the ma of the eca with an oa variant and mma branch variations [figures 46]. Of particular interest in this case was that the oa variant had two intra - orbital parts, namely an orbital division with a distinct branching pattern to supply the nasal cavity and nose (i.e. Ethmoidal and nasal branches) and an ocular division with a distinct branching pattern to supply the eyeball (i.e. Ciliary and muscular branches) and the retina (i.e. The central retinal artery as witnessed by choroidal blush). The orbital and ocular divisions coursed through the superior orbital fissure and optic foramen, respectively, according to expectation . The mma presented with two branch variations that originated from the anterior branch of the mma, namely a lacrimal branch that coursed through a cranio - orbital foramen and a meningeal branch that remained outside the orbit . Twelve - year - old boy presented with nasopharyngeal angiofibroma diagnosed with unilateral meningo - ophthalmic artery anomaly . Left lateral view angiogram of the head shows internal carotid artery (black arrow) with no ophthalmic artery . Twelve - year - old boy presented with nasopharyngeal angiofi broma diagnosed with unilateral meningo - ophthalmic artery anomaly . Left anteroposterior view angiogram of head shows meningo - ophthalmic artery anomaly (black arrow). (1: ophthalmic artery variant; 2: ocular division; 3: orbital division; 4: middle meningeal artery; 5: anterior branch; 6: meningeal branch; 7: lacrimal branch). Twelve - year - old boy presented with nasopharyngeal angiofibroma diagnosed with unilateral meningo - ophthalmic artery anomaly . Left view angiogram of head shows meningo - ophthalmic artery anomaly (black arrow). (1: ophthalmic artery variant; 2: middle meningeal artery; 3: anterior branch; 4: meningeal branch; 5: lacrimal branch). Nasopharyngeal angiofibroma often originates in the vicinity of the sphenopalatine foramen and epistaxis is a symptom to be reckoned with . Severe intra - operative bleeding in the current case is attributed to the presence of a rare vidian artery that is often encountered in nasopharyngeal angiofibroma . A vidian artery (i.e. The artery of the pterygoid canal) usually arises from the distal ma, or may have origin from the petrous ica (30% incidence), or may serve as anastomosis between the eca and the ica . Regardless of origin, a vidian artery participates in two major vascular networks through anastomoses in the pterygopalatine fossa (with pharyngeal, ethmoidal, and sphenopalatine arteries) and on the pharyngeal mucosa (with ascending pharyngeal and meningeal arteries), as witnessed in this patient . The presence of an inferolateral trunkus (ilt) with origin from the cavernous ica (c4 segment) and its anastomotic connections with the ma, mma, and oa must also be taken into consideration . An ilt is often identifiable in the dissection specimen, but is less frequently seen on imaging and was not witnessed in this case . Embryological explanations for m - oa and mma orbital routes are fetal anastomotic connections between the ica and eca and enlargement of fetal anastomosis between branches of the mma and oa, respectively . In principle, the stapedial system plays a pivotal role in the development of m - oa and mma orbital routes . Suggested embryonic evolutionary changes for the orbital collateral routes (m - oa and mma) encountered in this case are as follows: regression of the proximal parts of the stapedial artery (sa) (origin: ica) and primitive ophthalmic artery (poa) (origin: ica) occurred, and an annex between the distal parts of the sa and poa formed, whereby the m - oa gained eca origin . (1: internal carotid artery; 2: ophthalmic artery; 3: external carotid artery; 4: middle meningeal artery; 5: orbital branch; 6: stapedial artery; 7: maxillary artery; 8: meningo - ophthalmic artery; 9: meningeal branch; 10: lacrimal branch). Upon decision - making whether the m - oa and mma routes were tumor - related or incidental, it was evident that the latter was applicable in this case . In favor of the incidental presence of the m - oa and mma routes were their unilateral (left side) presence, the prominent right - side occurrence of the tumor, and no intra - orbital infiltrations . Of clinical significance is that in cases where the m - oa is the sole supply to the eye, proximal embolization of the ma or mma carries a high risk of visual impairment and must be avoided . A pre - operative routine angiographic study in a patient who presented with a nasopharyngeal angiofibroma revealed an m - oa anomaly . The m - oa anomaly followed distinct patterns of orbital supply and was the sole supply to the eye . In this particular case, the m - oa anomaly was not tumor related and was considered incidental . Proximal embolization of the ma was considered unsafe for tumor extirpation, but embolization of both sphenopalatine arteries was a useful tool in the management of epistaxis . Documentation of this tumor entity and m - oa anomaly, both relatively rare, remains a valuable contribution to the literature.
There was no overlap of individuals between the three studies, thus allowing independent confirmation of findings . On the basis of the danish central person registration number, the copenhagen city heart study (cchs, n = 9,121; recruited 19911994) (5) and the copenhagen general population study (cgps, n = 24,195; recruited 20032007) (6) recruited participants randomly from the general population of copenhagen, denmark . The studies included a self - administered questionnaire, a physical examination, and blood samples . We recruited (20012007) consecutively 6,129 population - based patients with diabetes from copenhagen county who attended the steno diabetes centre . Transferrin saturation (percentage) was determined as iron levels (in micromoles per liter) divided by 2 transferrin levels (in micromoles per liter) 100 . Transferrin was measured by turbidimetry, and iron levels were measured by colorimetry using a konelab autoanalyzer (thermo fisher scientific, waltham, ma) and hitachi 912 (roche diagnostics, indianapolis, in). Transferrin saturation> 50% was chosen as suggestive of increased transferrin saturation, in accordance with accepted clinical practice (79). Transferrin saturation range was 2.8236% (cchs), 0.9130% (cgps), and 0.197% (population - based case - control study). Increased transferrin saturation was detected in 355, 346, and 361 individuals in the cchs, cgps, and in the population - based case - control study, respectively . A combination of icd codes, self - reported diabetes (yes / no), information on antidiabetic medication, and a nonfasting glucose> 11 mmol / l was used . Cox proportional hazards regression with age as timescale (i.e., left truncation) was used to estimate hazard ratios (hrs) with 95% cis . The assumption of proportional hazards was tested with the use of schoenfeld residuals, and no violations were observed . Analyses not stratified for sex were adjusted for sex . In the case - control study, conditional logistic regression models were used to estimate odds ratios (ors) with 95% ci . The investigation was conducted according to the principles outlined in the declaration of helsinki . Written informed consent there was no overlap of individuals between the three studies, thus allowing independent confirmation of findings . On the basis of the danish central person registration number, the copenhagen city heart study (cchs, n = 9,121; recruited 19911994) (5) and the copenhagen general population study (cgps, n = 24,195; recruited 20032007) (6) recruited participants randomly from the general population of copenhagen, denmark . The studies included a self - administered questionnaire, a physical examination, and blood samples . We recruited (20012007) consecutively 6,129 population - based patients with diabetes from copenhagen county who attended the steno diabetes centre . Transferrin saturation (percentage) was determined as iron levels (in micromoles per liter) divided by 2 transferrin levels (in micromoles per liter) 100 . Transferrin was measured by turbidimetry, and iron levels were measured by colorimetry using a konelab autoanalyzer (thermo fisher scientific, waltham, ma) and hitachi 912 (roche diagnostics, indianapolis, in). Transferrin saturation> 50% was chosen as suggestive of increased transferrin saturation, in accordance with accepted clinical practice (79). Transferrin saturation range was 2.8236% (cchs), 0.9130% (cgps), and 0.197% (population - based case - control study). Increased transferrin saturation was detected in 355, 346, and 361 individuals in the cchs, cgps, and in the population - based case - control study, respectively . A combination of icd codes, self - reported diabetes (yes / no), information on antidiabetic medication, and a nonfasting glucose> 11 mmol / l was used . Cox proportional hazards regression with age as timescale (i.e., left truncation) was used to estimate hazard ratios (hrs) with 95% cis . The assumption of proportional hazards was tested with the use of schoenfeld residuals, and no violations were observed . Analyses not stratified for sex were adjusted for sex . In the case - control study, conditional logistic regression models were used to estimate odds ratios (ors) with 95% ci . Details are provided in the supplementary data online . The hrs (95% ci) for any form of diabetes in individuals with transferrin saturation of 50% vs. <50% were 1.8 (1.42.4; p = 0.001) in cchs and 1.5 (1.02.1; p = 0.03) in cgps (fig . The or (95% ci) for any form of diabetes in individuals with transferrin saturation of 50% vs. <50% was 3.3 (2.64.2; p = 0.0001). In the combined studies, or in the meta - analysis under the random - effects model was 2.1 (1.33.4; p = 0.003) for any form of diabetes in those with transferrin saturation 50% vs. <50% (fig . The ors for type 1 and type 2 diabetes in the combined studies in the meta - analysis under the random - effects model were 2.6 (1.25.6; p = 0.01) and 1.7 (1.42.1; p = 0.001), respectively, in those with transferrin saturation 50% vs. <50% (fig . 1b and c); statistical heterogeneity was q = 15 and p = 0.001 for type 1 diabetes and q = 0.7 and p = 0.7 for type 2 diabetes . Risk of diabetes according to transferrin saturation 50% vs. <50% in the cchs, the cgps, a case - control study, and combined . The case - control study comprised patients with diabetes from the steno diabetes centre in copenhagen, and control subjects were ascertained as in the cgps but from a different sample than those included in the cgps - only study . Combined, there was no overlap of individuals among these three studies, thus allowing independent confirmation of findings . Heterogeneity for the combined results for men and women was q = 17 and p = 0.001 for any form of diabetes, q = 15 and p = 0.001 for type 1 diabetes, and q = 0.7 and p = 0.7 for type 2 diabetes . We demonstrated that transferrin saturation 50% was associated with a two- to threefold increased risk of developing any form of diabetes, as well as type 1 diabetes and type 2 diabetes separately . This is the first and largest population - based study that consistently demonstrates transferrin saturation as a risk marker of any form of diabetes, and of type 1 and type 2 diabetes separately, in three independent studies . There was no overlap of individuals between the three studies, thus allowing independent confirmation of findings . Because the risk of developing diabetes observed in this study increased with the degree of iron overload for which there is a simple treatment (phlebotomy), 13% of diabetes cases in the general population and 7% of cases in a diabetes population likely could be resolved if patients received such a treatment earlier in life, equal to 300 patients / million in denmark . These findings reinforce the importance of investigating iron overload in the differential diagnosis of secondary causes of diabetes and support arguments in favor of screening for iron overload in the general population . A health - economic analysis of the cost / benefit ratio of screening for elevated iron saturation in the general or in diabetic populations may help to determine the public health consequences of this study (e.g., advice against iron supplements), and whether large prospective intervention studies should be initiated to show causality between elevated transferrin saturation and diabetes.
To investigate the immunologic events underlying the evolution of a chronic colitis we analyzed the chronic tnbs- colitis occurring in balb / c mice administered weekly doses of intra - rectal trinitrobenzene sulfonic acid (tnbs) (6, 7). Mice treated in this way initially develop intense colitis associated with severe weight loss and considerable mortality . However, about three weeks after the initiation of this form of tnbs - colitis, the colitis moderates and, while the mice do not exhibit the weight gain of control mice, they regain their lost weight . Importantly, the termination phase of this inflammatory cycle is accompanied by the development of fibrotic cycle . Thus, four - five weeks after initiation of chronic tnbs - colitis, the mice develop steadily increasing fibrosis of the colonic lamina propria . As shown in figure 1, this increase in intestinal fibrosis can be demonstrated by measuring the collagen content of the colon . To understand the immunologic basis of this complex series of events we determined the cytokines produced by lamina propria cells during the various stages of the inflammation . As established as an overview in figure 2, we noted that the production of th1 cytokines, il-12p70 and ifn- by extracted cells were greatly elevated on day 7 after initiation of tnbs administration . However, surprisingly, the production of these cytokines gradually decreased over the next 2 weeks, returning to baseline levels by day 21 and then gradually declining to levels increasingly below that observed at baseline . Interestingly, an increased production of an alternative set of pro - inflammatory cytokines, namely il-23 and il-17, accompanied the decline in the production of il-12p70 and ifn-. Increased il-23 production above baseline was noted first on day 21, and thereafter increased further until it reached a plateau on day 42 . The expression levels of both cytokines increased further until they reached a plateau on day 42 . Somewhat surprisingly, increased production of il-23 and il-17 coincided with increased production of cytokines usually considered a th2 cytokines, namely, il-25 and il-13 . Previous studies have shown that il-25, a member of the il-17 family of proteins (and designated il-17e), is associated with expression of th2 cytokines, at least when it is over - expressed in transgenic mice (8). On day 35 a massive increase in il-25 secretion appeared and thereafter il-25 secretion further increased, reaching its highest level on day 49 . At about the same time, the secretion of il-13 was initiated and the level of this cytokine also increased until it reached a plateau on day 49 . Coming now to the molecular events during intestinal fibrogenesis, in previous studies we showed that il-13 induces tgf-1 production by signaling through cell - surface il-13r2, an il-13 receptor formerly thought to function only as a soluble decoy receptor (912). Since il-13 is produced in the later phase of chronic tnbs - colitis we reasoned that this pathway might be involved in the development of fibrosis occurring during this phase . To explore this possibility we first determined the expression of both il-13r1 and il-13r2 in extracts of colonic lpmc during the course of chronic tnbs colitis . As shown in figure 3, il-13r1 was constitutively expressed throughout the time course of the animal model . In contrast, il-13r2 was an induced receptor since it was not evident in nave mice or in the first few weeks of chronic tnbs - colitis, but could be detected initially at a low concentration on day 35 and later at a high concentration on days 42 and 49 after initiation of colitis . The above time course of il-13r2 expression correlated with that of il-13 and was thus consistent with the possibility that the latter was an inducing cytokine . To address this question more directly we next blocked il-13 activity by administration of a plasmid (pci - sil-13r2-fc) encoding a soluble il-13r2-fc fusion protein (pci - sil-13r2-fc) or a control plasmid (pcl empty vector). The administration of the pcl - sil-13r2-fc plasmid by this route, but not the control plasmid, led to a reduction in lpmc production of il-13 . This effect is consistent with that noted previously in a study by chiaramonte et al where it was shown that mice with il-13r2 receptor deficiency have reduced il-13 production and is a possible result of the fact that autocrine il-13 signaling via this receptor is necessary for optimal il-13 production (9). In addition, as shown in figure 4, administration pci - sil-13r2-fc led to virtually complete loss of il-13r2 expression in extracts of colonic tissue obtained at the conclusion of the study (day 49). In contrast, neutralization of tgf-1 by administration of anti - tgf-1 had no effect on receptor expression or signaling; this was expected since there is no evidence to suggest that tgf-1 induces up - regulation of il-13r2 . Taken together, these results strongly suggest that il-13 production occurring in the late phase of chronic tnbs - colitis is indeed responsible for the expression of il-13r2 . In previous studies we have shown that intra - rectal administration of nf-b decoy oligonucleotides on days 35 and 42 abrogates the inflammation and the accompanying cytokine response characteristic of the chronic inflammation at this stage of the inflammation, including the high levels of il-23, il-17 and il-13 (6). In addition, such treatment prevented the production of tgf-1 and the development of intestinal tissue fibrosis . However, while these data established that the il-13 and tgf-1 responses as well as the collagen deposition were an outgrowth of the inflammation, they did not address the question of whether or not il-13 and/or tgf-1 were the cause of such deposition . To address the question of whether il-13 and tgf-1 are responsible for the collagen deposition, in the present study we determined the effect of in vivo inhibition of either il-13 signaling or tgf-1 activity on the generation of tissue fibrosis . In these studies we inhibited il-13 signaling in two ways: 1) by intra - nasal administration of the plasmid described above encoding a soluble il-13r2-fc fusion protein (pci - sil-13r2-fc) or a control plasmid (pcl empty vector) to block the receptor with the use of a decoy; and 2) by intra - rectal administration of il-13r2-specific sirna encapsulated in hvj - e or a control sirna to down - regulate the receptor by gene silencing . Both the sil-13r2-fc plasmid and the sirna were administered every other day starting on day 35 after the initiation of the chronic inflammation . On the other hand, for study of the effects of inhibition of tgf-1 we administered anti - tgf--antibody or isotype control ig by an intra - peritoneal route over the same time period . The effects of these treatments on il-13 and tgf-1 levels were subsequently determined at the termination of the study on day 49 . For this purpose, lpmc extracted from the colons of sacrificed mice were cultured for 48h with anti - cd3/anti - cd28 and their secretion of il-13 and tgf-1 into the culture supernatant measured by elisa . Neutralization of il-13 signaling by fusion protein or gene - silencing of il-13r2 by sirna as well as neutralization of tgf-1 by antibody was accompanied by only a small, non - significant increase in body weight and no measurable change in colitis score . Thus, lpmc extracted from the colons of sacrificed mice on day 49 and evaluated for secretion of cytokines secreted equal amounts of il-12p70, ifn-, tnf-, il-23, il-17, il-25 and il-4 whether or not they were subjected to neutralization of il-13 signaling or tgf-1 . In contrast, a very different picture was obtained with respect to tgf-1 secretion . In this case, the cells extracted from the colons of mice subjected to neutralization of il-13 signaling by fusion protein or il-13r2 downregulation by sirna exhibited greatly reduced production of tgf-1 . In addition, as shown in figure 5, those mice subjected to neutralization of il-13 signaling as well as those subjected to tgf-1 neutralization displayed basal collagen levels seen in nave mice whereas those mice not subjected to such neutralization displayed undiminished collagen levels as compared to other mice with chronic tnbs - colitis . Overall, these data show quite clearly that neutralization of il-13 signaling via il-13r2 does not alter the course of the inflammation of chronic tnbs - colitis but does block the development of fibrosis; in addition, they show that such signaling results in fibrosis via its effect on the induction of tgf-1 . In summary, these findings suggest that chronic inflammation is orchestrated by a succession of cytokines that ultimately result in il-13 and tgf-1 production . In addition, blockade of this cytokine interaction can prevent intestinal fibrosis by selectively interfering with the pro - fibrotic program.
Integral membrane proteins account for 2030% of fully sequenced proteomes (1). To date, two major structural architectures can be distinguished: the -helical and the -barrel membrane proteins . The former are located primarily in cell membranes of eukaryotic cells and bacterial inner membranes, while the latter are found exclusively in the outer membranes of gram - negative bacteria and in the outer membranes of mitochondria and chloroplasts (24). The -barrel outer membrane proteins (omps) are crucial for the life of bacteria, serving a variety of diverse roles, such as passive nutrient uptake and active transport of large molecules, protein secretion, enzymatic activity or adhesion to host cells (58). The difficulty in obtaining crystals suitable for high - resolution studies of outer membrane proteins has resulted in their under - representation in the protein data bank (9) (<1% of all deposited proteins with known 3d structure). Given these facts, and because many -barrel omps nowadays attract an increased medical interest, several approaches have been made towards the development of predictive algorithms for this type of proteins . These methods are based grossly on hydrophobicity (10) and statistical analysis (11,12), remote homology detection (13), hidden markov models (hmms) (1418), feed - forward neural networks (1921), radial basis function neural networks (22,23) and support vector machines (24), whereas others like bomp (25), tmb - hunt (26,27) and the tmb - finding pipeline (28) are oriented towards genome scale discrimination of -barrel membrane proteins . A previously presented benchmark of several topology prediction methods indicated that hmms are the most reliable predictors (29). Special purpose biological databases that include certain families of -barrel proteins, like tcdb (30), pdbtm (31), topdb (32), psortdb (33), tmbeta - genome (34), opm (35), mptopo (36), membrane protein data bank (37), prdns (38), tmpdb (39), tmfunction (40) and the hhomp database, as part of the hhomp webserver (13) have also been available to the public . However, the annotation and classification of -barrels in the aforementioned databases is incomplete, the coverage is limited and there are also many false positives (i.e. Lipoproteins or peripheral proteins); this urges the need for intensive studies and careful data collection regarding these proteins . In order to create ompdb, we based our research along three main axes, namely the available 3d structures, the profile hmms (phmms) deposited in version 24.0 of the pfam database (41) that correspond to domains found solely in transmembrane (tm) -barrel omps, coupled with an extensive literature search for novel -barrel proteins that are reported as such but cannot be retrieved from the databases otherwise . The ultimate purpose was to build a classification system where each family (i.e. A -barrel domain) would be represented by a unique phmm . We should emphasize here, that tm -barrel proteins could possess either a single - domain architecture (where the whole sequence is composed of the -barrel domain), or a multi - domain architecture (in which case the tm -barrel domain comprises only a portion of the sequence). Thus, following the philosophy of domain databases such as pfam, we identified only the respective domains that are responsible for the -barrel formation and subsequently we included in the database only proteins possessing these domains . Initially, we retrieved all proteins with a known 3d structure that are deposited in pdb and are listed at stephen white s laboratory page at uc irvine (http://blanco.biomol.uci.edu/membrane_proteins_xtal.html). We chose to exclude -barrels that do not originate from gram - negative bacteria, such as the mitochondrial voltage - dependent anion channel (pdb i d: 2jk4) (42), the mspa mycobacterial outer membrane channel (pdb i d: 1uun) (43) or the -hemolysin (pdb i d: 7ahl) (44) and lukf (pdb id:3lkf) (45) from staphylococcus aureus . We studied the outer membrane -barrel protein superfamily clan (mbb; cl0193) of the pfam database which contains 36 members and noticed that there were several tm -barrel proteins with crystallographically solved structure whose respective pfam domains were not included in the clan . Such examples include scry (pdb id:1a0s), ompla (pdb id:1fw2) and pagp (pdb id:1mm4), which correspond to pfam domains pf02264, pf02253 and pf07017, respectively . Salmonella typhimurium lpxr (pdb id:3fid) had a hit in a pfam domain which was neither included in the clan nor had any description for its function (pf09982). Finally, two well - studied porins from rhodobacter capsulatus (pdb id:2por) and rhodopeudomonas blastica (pdb id:1prn) had no hits in the pfam domains . Apart from that, by searching in the literature, we came up with a number of proteins that were experimentally characterized as -barrel (either multi- or single - domain) omps using several techniques (i.e. Sub - cellular fractionization, electron microscopy, protease protection experiments, channel properties, chemical labeling, heat modifiable activity and so on). Many of them had either no hit in the phmms of pfam or matched any of the automatically generated alignments in the pfam - b subset . For these proteins there was also no evidence concerning their sub - cellular location or structure in the uniprot database (46). In the first case, we performed a blast search (47) against uniprot using as query the -barrel domain of the experimentally verified protein(s) reported in the respective published reports, we created multiple alignments with the best - scoring results using clustalw (48) and then, we built phmms for the particular domain using version 3.0b3 of the hmmer (49) software package . In the second case, we constructed the phmms based on the domain alignments deposited in pfam - b after screening them in order to remove possible partial hits (sequence fragments). Finally, we used the total set of phmms that was created for retrieving additional proteins from uniprot that scored high in these phmms, indicating that they could be classified in one of the available families . These proteins were appended in the multiple alignments representing the respective -barrel domains and the procedure was repeated until no new members could be found . The procedure for creating the phmm for the newly identified -barrel domains is illustrated graphically in figure 1 . We have to notice, that some very short protein sequences (i.e. Less than100 amino acids) that scored high against the phmms, were removed from the database since they could not possibly fold into a -barrel structure . Figure 1.schematic ilustration of the procedure used to create the phmms that represent the tm domains of -barrel omps . Either we were dealing with (a) single - domain omps or (b) multi - domain ones, following the literature we isolated the domain responsible for the -barrel formation . Afterwards, we performed a blast search (47) against uniprot using as query the -barrel domain of the experimentally verified protein reported in the respective published reports, we created multiple alignments with the best - scoring results using clustalw (48) and then, we built phmms for the particular domain using version 3.0b3 of the hmmer (49) software package . The phmm was subsequently used to search for new family members and the procedure iterated until no new family members are found . For families that were represented in pfam - b subset, schematic ilustration of the procedure used to create the phmms that represent the tm domains of -barrel omps . Either we were dealing with (a) single - domain omps or (b) multi - domain ones, following the literature we isolated the domain responsible for the -barrel formation . Afterwards, we performed a blast search (47) against uniprot using as query the -barrel domain of the experimentally verified protein reported in the respective published reports, we created multiple alignments with the best - scoring results using clustalw (48) and then, we built phmms for the particular domain using version 3.0b3 of the hmmer (49) software package . The phmm was subsequently used to search for new family members and the procedure iterated until no new family members are found . For families that were represented in pfam - b subset the majority of proteins that we collected would not be possible to be gathered by just searching in uniprot using a combination of keywords, because they were mostly listed as having the result of the aforementioned procedure was a total of 85 families and 84 phmms (one family has only one representative, thus no multiple alignment and, consequently, no phmm model could be created). At the end, we performed once again a literature search in order to find additional references for all the -barrel families (i.e. Domains) that we included in the database . Another main innovation of ompdb is the annotation of the tm segments . For proteins with known 3d structure, we used the information provided in the pdbtm database regarding the annotation of -barrels along with the number and location of -strands . For proteins that share structural homology with a protein with determined 3d structure (i.e. They belong to the same family), we mapped the boundaries of the tm segments of the member with the crystallographically solved structure to their sequences, using the alignment of the family s sequences . Some obvious errors, such as a porin with 17 tm strands, were manually corrected . For families for which there was no evidence for tm topology, we offer the user the opportunity to run the pred - tmbb algorithm, which is one of the top - scoring algorithms concerning -barrel omps prediction (14,15,29). Ompdb, in its first version, contains 69 354 -barrel outer membrane proteins, originating from 2712 gram - negative bacterial species and strains, which are classified into 85 families . Figure 2 summarizes the annotation regarding the families classification system we propose in the pfam database, which is the largest collection of protein families in the literature . However, if we just relied on the mbb clan, we would end up with only 36 profile hmm models that are identified by the curators of the database as being characteristic for -barrels . A more detailed analysis, initiated by literature findings, revealed another 22 models deposited in pfam - a, the high quality, manually curated component of pfam, which represent -barrels . Four out of them were actually assigned as domains of unknown function (dufs), which lack further characterization in pfam . We also used nine multiple sequence alignments (msas) from the automatically generated pfam - b subset, because some of the proteins they contained were found to be -barrels during the literature search . Both the 22 pfam - a models and the nine msas from pfam - b are not straightforward to be retrieved from uniprot or pfam just by using some kind of keywords combination in a text search query, since in most cases such keywords do not exist in the respective entries . More importantly, for 17 of ompdb s families, there was no information in the pfam database at all . Figure 3 shows a summary of the database s families and proteins entries classified according to their function . Figure 2.annotation of ompdb s families in the latest version (v.24october 2009) of the pfam database . The majority of the families (36) is reported in the mbb clan part of the pfam database . However, many phmm models correspond to domains that represent families not included in the clan (18 + 4 = 22 families) or they can only be found in the non - annotated pfam - b subset of pfam (nine phmms). Finally, out of the 84 phmm models that ompdb contains, 17 were not reported in pfam at all and these correspond to novel families . Figure 3.a demonstration of the total numbers of families and protein entries in ompdb that belong to each of the eight functional categories that we created . There are still a lot of families with -barrel proteins whose function remains unclear, whereas specific and non - specific channels, together with biogenesis / secretion proteins seem to play a vital role in the bacterial life . Receptors are under - represented in terms of the number of families, but they constitute the second largest category of -barrel omps in absolute numbers of proteins . In general, the largest proportion of outer membrane proteins in gram - negative bacteria serve as receptors or play a role in biogenesis / secretion . The category of proteins with unknown function, though it contains a lot of families, has a relatively small number of protein members, which may be due to the progress in whole genome annotation that is being made regarding gram - negative bacteria . Annotation of ompdb s families in the latest version (v.24october 2009) of the pfam database . The majority of the families (36) is reported in the mbb clan part of the pfam database . However, many phmm models correspond to domains that represent families not included in the clan (18 + 4 = 22 families) or they can only be found in the non - annotated pfam - b subset of pfam (nine phmms). Finally, out of the 84 phmm models that ompdb contains, 17 were not reported in pfam at all and these correspond to novel families . A demonstration of the total numbers of families and protein entries in ompdb that belong to each of the eight functional categories that we created . There are still a lot of families with -barrel proteins whose function remains unclear, whereas specific and non - specific channels, together with biogenesis / secretion proteins seem to play a vital role in the bacterial life . Receptors are under - represented in terms of the number of families, but they constitute the second largest category of -barrel omps in absolute numbers of proteins . In general, the largest proportion of outer membrane proteins in gram - negative bacteria serve as receptors or play a role in biogenesis / secretion . The category of proteins with unknown function, though it contains a lot of families, has a relatively small number of protein members, which may be due to the progress in whole genome annotation that is being made regarding gram - negative bacteria . The database possesses a user - friendly environment, through which the user may retrieve all the necessary information or find available resources and cross - references . The web application is based on the combination of two layers: the underlying level is a mysql database system, which contains all protein data and the upper layer is an apache - php applications server that receives user queries and fetches populated html data to the web browser client . From the welcome page of the web site, the user can view a brief description of ompdb, along with the current holdings . A selection of a family entry from the drop down menu in the same page redirects to the respective family page . There, a short description of the family is presented along with the literature references, the initial (seed) and full alignments of the family s members (figure 4) the user can read a short description for the given family together with a number of respective literature references . By clicking on the respective web links, he / she can view all proteins that belong to the family or download the seed and full alignments of the family s protein members . The user can read a short description for the given family together with a number of respective literature references . By clicking on the respective web links, he / she can view all proteins that belong to the family or download the seed and full alignments of the family s protein members . From the navigation bar in every web page, there is a text search page, in which several fields are presented to the user, allowing the performance of advanced text search queries . There is also the opportunity to submit blast searches against the database s protein members using the blast search tool, where an e - value cut - off level can be specified accordingly . Additionally, through the domain search tool, one or more fasta - formatted sequences may be submitted and searched against the collection of profile hmms that was constructed and is proven to be characteristic for -barrel omps . This search can rely on either an e - value cut - off level or the manually specified trusted cut - off scores of each model . The results in all cases are presented in a tabular way, which facilitates easy view of the main fields of each result entry and can be selected and downloaded locally for further investigation . Each database entry contains the following fields: ompdb name, ompdb i d, uniprot accession number, protein description and classification, sequence, species, organism name, taxonomy, links to other databases, accompanied with annotation for tm segments and signal peptides (figure 5). There is also an extensive user s manual page, describing in detail the available tools . The user may observe the classification of the protein, the available cross - references, the amino acid sequence, along with information about the presence of a signal peptide and the annotation of the tm segments . All this information can be downloaded in easy - to - use formats as well (fasta, xml or text files) the user may observe the classification of the protein, the available cross - references, the amino acid sequence, along with information about the presence of a signal peptide and the annotation of the tm segments . All this information can be downloaded in easy - to - use formats as well (fasta, xml or text files). We have constructed a relational protein database, which contains -barrel outer membrane proteins from gram - negative bacteria . To our knowledge, ompdb has some innovative and unique features not available in any other publicly accessible resource . When compared to general protein or domain databases, like uniprot and pfam (which were actually the main sources of information), ompdb presents a more complete classification and accurate annotation concerning -barrel proteins, due to the added value of the manual annotation that we performed and the detailed literature references that we provide . On the other hand, a comparison of ompdb against other specific databases that contain -barrel proteins, reveals that it clearly excels at all aspects, because it features the largest number of protein and family entries, it possesses the most complete and exclusive data for -barrels and offers the most complete interconnection to other public databases, literature references, prediction tools and sequence annotation . Table 1 shows a summary of the comparison of the main characteristics of ompdb as opposed to other related databases that are available to the scientific community . From the table, it is evident that related databases fall into two main categories: there are those with a very limited number of protein entries and either no classification system for them or families with just few representative proteins . The reason for this is that they contain only proteins that have a crystallographically (3d) determined structured (like tcdb, pdb_tm, topdb, opm, mpdb and tmpdb). On the other hand, there are databases like tmbeta - genome, prnds and psortdb that contain a larger number of proteins, but they offer no classification system and additionally, the proteins are gathered using automated techniques (i.e. Prediction algorithms). We should note here, that prediction methods produce a large number of false positives and thus, the content of these databases need additional filtering (i.e. They contain several outer membrane lipoproteins or peripheral proteins). It is clear thus, that the semi - automated procedure used in the development of ompdb, combines the advantages of both approaches offering currently the most advanced resource for omps . Table 1.a comparison of ompdb to other databases that include -barrel outer membrane proteins, in terms of total entries, method of data retrieval, existence of literature references, annotation of important sequence features (such as the signal peptide and the tm segments), interconnection to other public databases related to -barrel proteins and availability of prediction / search toolsdatabase featuresompdb (current work)pfam (mbb clan) (41)tcdb (30)pdb_tm (nr set) (31)topdb (32)epsortdb (33)tmbeta genome (34)opm (35)mpdb (37)prnds (38)tmpdb (39)hhompdb (13)total entries69 35438 595348494548036 0957314831821754 184total families8536572625data retrievalsemi- automatedsemi- automatedmanualsemi- automatedsemi- automatedmanualautomatedsemi- automatedmanualautomatedmanualautomatedliterature references+++++++signal peptide/ tm segments annotation+/+///++/+///+//+/+/number of databases cross - referenced13511223223prediction / search toolsblast/ hmmer/ pred - tmbb/ text searchtext search/ hmmertext search/ blasttext searchtext searchpsortb/ blastsearch by organismtext searchtext searchtext search/ bss / bbftext searchhhsearch a comparison of ompdb to other databases that include -barrel outer membrane proteins, in terms of total entries, method of data retrieval, existence of literature references, annotation of important sequence features (such as the signal peptide and the tm segments), interconnection to other public databases related to -barrel proteins and availability of prediction / search tools ompdb aims to fill a gap in the literature, because as already mentioned, the annotation of tm -barrels in the existing databases is rather inadequate . Experimentalists that are involved with the biochemical and functional characterization of outer membrane proteins of gram - negative bacteria will probably benefit the most from our database . For instance, a blast or domain search query against ompdb, which would follow the identification of one or more -barrel protein sequences, is expected to be much more informative than a usual query against uniprot, the non - redundant (nr) sequences of ncbi or pfam . Moreover, even in the case that a similarity (even a remote one) is not found, the existing prediction methods that have been developed in our lab (pred - tmbb and conbbpred), will provide the necessary tools towards the clarification of the structural and functional nature of these proteins . Another aspect in which ompdb could be very useful is the computational and theoretical analyses concerning -barrels . The classification into families might be used in studies regarding the attributes of their members, in phylogenetic analyses and/or comparative genomics . The use of the phmms that are deposited in the database could also be used for designing effective strategies for whole - genome analyses . Finally, the existence of such a large and reliable data set of -barrels can be used for large - scale analyses concerning the classification accuracy of existing predictors, for training new prediction methods or for comparative modeling approaches . We permit the users to download the entire database, in various easy - to - use formats, for those bioinformaticians and/or experimentalists who would like to access a frequently updated, high - quality annotationed data set of -barrel outer membrane proteins . We also used the cd - hit program (50) in order to provide nr subsets of the database at various levels, i.e. 30% (4225 proteins), 50% (15 029 proteins), 70% (23 110 proteins) and 90% (31 327 proteins) sequence similarity . The database may also be useful for microbiologists whose aim is to identify potential targets in bacterial genomes for medical diagnostics, vaccines, antimicrobials and other uses . Our long - term goal is to keep ompdb as up - to - date as possible, following the regular updates of uniprot and searching in the literature at the same time, for novel, experimentally verified -barrel proteins, in order to include them in the database or appoint them to a new family if necessary . Similar to other databases, ompdb is an ongoing project and interaction with the user community is vital for its development and refinement . We encourage the submission of data, correction of errors, and suggestions for making ompdb of greater use to the scientific community.
The centers for disease control projects that more than half of hiv-1 infected individuals in the united states will be over the age 50 by the year 2015 . The increasing life expectancy of hiv-1 infected patients has resulted in an unexpected increase in aging - related co - morbidities, placing hiv - positive elders at increased risk of morbidity and mortality . One important example of this is with the newly described syndrome of frailty, which may play an important role in accelerated aging of hiv-1 infected adults . Frailty has been defined in the aged as a biologic syndrome of decreased reserve and resistance to stressors, resulting from the cumulative decline of physiologic systems and often progresses in a step - wise functional decline over time . The clinical importance of frailty is that the syndrome is considered a high - risk state, predictive of adverse health outcomes such as decreased function and mobility, hospitalization and death.numerous studies in the last 10 years have tried to assess frailty in different populations . Studied frailty in men and women older than 65 years of age who were enrolled in a cardiovascular study . Described a frail phenotype that even in the absence of disabilities or co - morbidities showed that 7% of population older than 65 years of age are frail whereas, 20 - 26% older than 80 years of age were frail . Frailty can be a primary finding, but also a secondary diagnosis as a result of an acute event or co - morbidity like malignancy, atherosclerosis, infection (hiv), or depression.additionally, other factors likely contribute to frailty in hiv patients, for example, intravenous drug abuse, indigence and mental illness . Frailty has been found in hiv-1 infected patients at younger ages than non - hiv infected patients . Older hiv-1 infected individuals frequently present with more severe hiv disease and have a shorter survival time than younger individuals, often because they are not diagnosed until very late in the disease process . Another reason may be that older patients have more co - morbid diseases interacting with hiv-1 . Older hiv-1 infected individuals have been described as frailer than age - matched control individuals without hiv-1 infection . Clinical measurement of frailty in hiv-1 infected patients is important as frailty may be reversible in its early stages (e.g. Interventions to reverse deconditioning, protein - energy malnutrition, depression, vitamin d deficiency and other frailty related conditions) before depleted reserves reach a critical threshold leading to irreversible vulnerability and functional decline . Because it is impractical to test for frailty in all patients attending an hiv clinic we recommend the following patients be assessed for the presence of frailty: patients entering care with a cd4 cell count <200, patients complaining of unintentional weight loss, severe neuropathy or patients who are not compliant with hiv therapy . Obtain verbal consent of the patient to undergo tests for frailty or " weakness " . Consent need not be written since everything that is performed is part of a normal physical examination . The examiner states the three words out loud.instruct the patient to draw the face of a clock, either on a blank sheet of paper or on a sheet with the clock circle already drawn on the page . After the patient puts the numbers on the clock face, ask him or her to draw the hands of the clock to read a specific time.ask the patient to repeat the 3 previously stated words . Give 1 point for each recalled word . Patients recalling none of the three words are classified as cognitively impaired (score = 0). Patients recalling all three words are classified as cognitively intact (score = 3) patients with intermediate word recall of 1 - 2 words are classified based on the clock draw test (abnormal = impaired; normal = intact).if the patient fails the mini - cog, the clinician should examine further for causes of confusion and/or delirium . Instruct the patient to draw the face of a clock, either on a blank sheet of paper or on a sheet with the clock circle already drawn on the page . After the patient puts the numbers on the clock face, ask him or her to draw the hands of the clock to read a specific time . Ask the patient to repeat the 3 previously stated words . Give 1 point for each recalled word . Patients recalling none of the three words are classified as cognitively impaired (score = 0). Patients recalling all three words are classified as cognitively intact (score = 3) patients with intermediate word recall of 1 - 2 words are classified based on the clock draw test (abnormal = impaired; normal = intact). If the patient fails the mini - cog, the clinician should examine further for causes of confusion and/or delirium . Someone who is frail may have unintentional weight loss of 10 pounds in the prior year . Someone who is frail has a decreased walking time as defined by a timed 15-foot walk test . Men with a height of <173 cm and women with a height <159 cm who walked 15 feet in> 7 sec are considered frail; men> 173 cm and women> 159 cm who walked 15 feet in> 6 sec are considered frail . Determine if the patient has weakness . Weakness is established when there is decreased grip strength measured by a dynamometer with the value adjusted for gender and body mass index (bmi). Men with a bmi <24 are considered frail if the grip strength (kg) is <29, for a bmi of 24.1 - 28, a man is frail if <30, for a bmi> 28 a man is frail if <32 . For women, a bmi of <23 is considered frail if the grip strength (kg) is <17, a bmi 23.1 - 26 is considered frail if <17.3, a bmi of 26.1 - 29 is considered frail if <18, and a bmi> 29 is considered frail if <21 . This is established by a weighted score of kilocalories expended per week measured by the minnesota leisure time activity questionnaire . Frailty is present when males use <383kcal / week, and females <270 kcal / week . This is self - reported by answering 2 questions from the center for epidemiologic studies depression scale . The questions asked are: how often in the last week did you feel: (a) that everything i did was an effort? ; or (b) i could not get going? Answers were: 0 = less than 1 day, 1 = 1 - 2 days, 2 = 3 - 4 days, 3 = most of the time . Answering 2 or 3 to either of these questions is a positive criterion for frailty . One hundred outpatient hiv-1-infected persons were prospectively tested for clinical markers of frailty to include shrinking weight, slowness in walking, decrease in grip strength, low activity, and exhaustion . Eighty - one patients were not frail when studied, 19 patients were frail at the initial assessment (table 1). The characteristics of the frail and non - frail patients were not significantly different except for a higher rate of hepatitis c and neuropathy in the frail group (p <0.05). Length of infection with hiv, cd4 count and hiv-1 rna viral load were also compared . As shown in table 2 the younger patients had a greater incidence of frailty and this was associated with low cd4 cell counts . Cd4 counts <200 cells / mm were associated with 9-fold increased odds of frailty relative to patients with a cd4 count> 350 cells / mm (odds ratio [or] 9.0, 95% confidence interval [ci] 2.1 - 44). Seven frail patients were measured 6 months later: 2 died refusing therapy, 4 were no longer frail, and 1 patient remained frail (table 3). From this data it appears that when patients took prescribed anti - retroviral therapy the cd4 cell counts improved along with an improvement in the general state of health of the patient . Finally, if the incidence of frailty in the study population is compared to the number of years of anti - retroviral therapy taken by the patients, the longer patients took anti - retroviral therapy, the less the incidence of frailty (p <0.05). This fact supports the early use of anti - retroviral therapy in hiv - infected patients which is the preferred approach in recent hiv treatment guidelines . We conclude that frailty is common in hiv outpatients and is associated with low cd4 counts more than with advancing age . Our data suggest that frailty is transient, especially in younger patients who may revert to their prefrail state unlike uninfected elderly individuals in whom a stepwise decline in function may occur . Frail patients were compared to non - frail hiv patients with respect to sex, age and comorbidities . The presence of neuropathy and infection with hepatitis c were significant differences between the frail and non - frail patients . The frail patients were significantly younger and with lower cd4 cell counts than non - frail patients . These patients all needed anti - retroviral therapy, were prescribed therapy and then retested for frailty 6 months later . Only one patient who took the prescribed anti - retroviral therapy was still frail 6 months later . Relationship between number of years of taking anti - retroviral therapy and the incidence of frailty (89 patients were taking anti - retroviral therapy). The longer patients took anti - retroviral therapy the less the incidence of frailty in that population (p <0.05). Frail patients were compared to non - frail hiv patients with respect to sex, age and comorbidities . The presence of neuropathy and infection with hepatitis c were significant differences between the frail and non - frail patients . The frail patients were significantly younger and with lower cd4 cell counts than non - frail patients . These patients all needed anti - retroviral therapy, were prescribed therapy and then retested for frailty 6 months later . Only one patient who took the prescribed anti - retroviral therapy was still frail 6 months later . Relationship between number of years of taking anti - retroviral therapy and the incidence of frailty (89 patients were taking anti - retroviral therapy). The longer patients took anti - retroviral therapy the less the incidence of frailty in that population (p <0.05). Previous studies of hiv and frailty: two retrospective studies by desquilbet et al . Assessed frailty in a cohort of men who have sex with men from the multicenter aids cohort studies (macs). Both studies used a shortened definition of frailty containing fewer criteria than did our study . The first study compared frailty in hiv-1 infected men in the pre - treatment era to a control group of hiv uninfected men . There were similar rates of frailty in hiv+ men older than 55 years and hiv- men older than 65 years; frailty was found to occur earlier in hiv-1 infected men . Our study had similar findings of an earlier occurrence of frailty phenotype, but we obtained higher rates of frailty compared with macs, possibly because our use of the full fried frailty criteria versus surrogate administrative data, but also because of the different population of patients in our study . Another study by desquilbet et al . Evaluated cd4 cell count and hiv viral load as predictors of frailty in hiv+ men and found that lower cd4 cell counts and viral loads of more than 50,000 copies of rna were significantly associated with frailty . Also the prevalence of frailty declined in the era of art . Despite differences in measuring frailty and in population characteristics our study concluded like desquilbet et al . That a low cd4 cell count is significantly associated with frailty and that patients on long - term art have less likelihood of developing frailty . Premature occurrence of prevalence of frailty, shorter duration of art, more co - morbidities and lower cd4 count were associated with frailty in both studies, but we did not find a strong association between psychiatric diagnosis and frailty . We found a positive relationship between length of art and not being frail (figure 1). Our findings of frailty in hiv patients: our initial hypothesis that age was not significantly important when measuring frailty of patients with low cd4 cell counts was confirmed . Frailty is likely more causally related to the inflammatory state and profound immunosuppression found in many patients with low cd4 cell counts . Many of these patients had a history of recently treated opportunistic infections . Because of these observations we propose that an active diagnosis of aids (cd4 cell count <200 cells/l) is a significant co - morbidity itself and significantly predisposes patients to being frail . All of our frail patients had at least one co - morbidity besides hiv itself . Our frail patients <50 years had significantly fewer co - morbidities than the frail population> 50 years, though in comparison, the younger people had a lower cd4 cell count . Our other hypothesis was that frailty may be temporary in younger patients with low cd4 cell counts and may revert when cd4 cell counts improve . We were limited by the low number of patients and this hypothesis could not be proven, but it was a likelyexplanation for the small number of patients in which reversal was demonstrated (table 1). This fact on its own would support the recommendations of starting art at higher cd4 counts and continuing art without any treatment breaks . We believe that the main reason by which length of art treatment was shown to be protective for frailty is that patients on long term treatment are more likely to have better control of co - morbidities as well as hiv and less likely to be frail . In conclusion we have observed an association between low cd4-cell counts and frailty, which is not affected by age, viral load or the presence of co - morbidities . Effective treatment with art plays a protective role against frailty, reinforcing the importance of effective art . Early implementation of art in the care of hiv patients may protect against frailty . Though not tested in our study, future research should address other interventions known to reverse frailty in the aged including treating deconditioning, protein - energy malnutrition, depression, and vitamin d deficiency.
In the past two decades, there has been tremendous growth of intensive care medicine in india . However, there are scant data on the organizational aspects, case mix and practice patterns in indian intensive care units (icus). Most of the available information comes from either single - center studies or studies in specific groups of patients or conditions . It is essential to have data from several indian icus to reflect the vast and diverse spectrum of critical care illness, services, and practices . Such information may be useful to identify deficiencies in the organization of care and to identify targets for education, clinical improvement, and research . The data would provide baseline estimates of disease prevalence, severity of illness and mortality, essential for designing research studies . This multicenter study was planned to gather such information about icus all over india . In order to enable participation from a large number of icus throughout the country, we used a point prevalence design, obtaining data on four different days over a 1-year period . This was a multicenter, observational, point prevalence study, performed on four separate days . All patients present in the icu on july 14, 2010, october 13, 2010, january 12, 2011 and april 13, 2011 were included in the study . These days were the second wednesdays in the 1 month of each quarter and were representative of the major seasons . Data were recorded for all patients present in the icu during the 24 h starting 08.00 am on the study day to 08.00 am next day . The first time an icu joined the study, data about the icu was recorded . A closed icu was defined as one in which final orders for the patient were written by the icu team; an open icu was defined as one in which care of the patient was directed by non - icu doctor teams, and orders could be written by non - icu team doctors . A center was considered adequately equipped if all the following facilities were available either in the icu or in the hospital: renal replacement therapy (rrt), computerized tomography scan, microbiology, biochemistry and hematology laboratories, echocardiography and cardiac catheterization laboratory . Facilities available in the intensive care unit and hospital in 120 centres intensive care unit characteristics data collected for each patient included the following: primary reasons for icu admission, source of admission, demographics, patient characteristics and comorbidities as listed in tables 3 and 4 . Admission was defined as surgical if the patient was admitted to the icu from the operation theatre or recovery room . Elective surgery was defined as a surgical procedure which was planned more than 24 h prior to icu admission . Emergency surgery was defined as a surgical procedure before icu admission which was planned <24 h in advance . The primary reason for icu admission was the single most applicable diagnostic category based on the acute physiology and chronic health evaluation (apache) iii classificationage, physiological parameters, and comorbidities used to calculate the apache ii score and sequential organ failure assessment (sofa) score . Physiological variables were the worst recorded values during the 24-h study period . When data for any parameter required calculation of the apache ii and sofa score was missing, that parameter was assumed to be normal . A sofa score of 3 or 4 for any individual organ was used to identify organ failureinterventions are listed in table 5presence of infection (suspected or proven infection at icu admission or during the 24-h study period); severe sepsis or septic shock (svspss) at any time during the patient's icu stay; suspected or proven tropical infections (malaria, dengue, leptospirosis, scrub typhus); microrganisms, and antibiotic therapy . Primary reasons for icu admission, source of admission, demographics, patient characteristics and comorbidities as listed in tables 3 and 4 . Admission was defined as surgical if the patient was admitted to the icu from the operation theatre or recovery room . Elective surgery was defined as a surgical procedure which was planned more than 24 h prior to icu admission . Emergency surgery was defined as a surgical procedure before icu admission which was planned <24 h in advance . The primary reason for icu admission was the single most applicable diagnostic category based on the acute physiology and chronic health evaluation (apache) iii classification age, physiological parameters, and comorbidities used to calculate the apache ii score and sequential organ failure assessment (sofa) score . When data for any parameter required calculation of the apache ii and sofa score was missing, that parameter was assumed to be normal . A sofa score of 3 or 4 for any individual organ was used to identify organ failure interventions are listed in table 5 presence of infection (suspected or proven infection at icu admission or during the 24-h study period); severe sepsis or septic shock (svspss) at any time during the patient's icu stay; suspected or proven tropical infections (malaria, dengue, leptospirosis, scrub typhus); microrganisms, and antibiotic therapy . Primary reason for intensive care unit admission patient demographics, intensive care units admission characteristics and severity of illness * survival status at icu discharge was recorded within 30 days from the day of the study . Patients discharged alive from icu were followed up till hospital discharge, or 30 days from the day of the study, whichever was earlier . For patients dying in the icu, investigators were asked to record whether any form of limitation of treatment occurred . Terminal discharges (tds) from icu to a location outside the hospital, either on family or patient request, as well as those documented as left against medical advice, were also recorded . Nonsurvivors were patients who died in the icu as well as those who were tds within 30 days from the day of the study . Secondary outcomes were as follows: hospital mortality, defined as a composite of those who died in the hospital and tds from the icu within 30 days from the day of the study, and icu and hospital lengths of stay, till 30 days from the study day . The standardized mortality ratio (smr) using the hospital mortality and that predicted by apache ii was calculated for those patients who were admitted to the icu within 24 h of the study day . For calculation of icu and hospital length of stay, missing data analysis was confined to data from adult patients (> 16 years of age). Continuous variables were compared with the use of the student's t - test, analysis of variance, mann - whitney test or the kruskal - wallis test . Multivariate binary logistic regression analysis was performed to determine the independent predictors of icu mortality . A stepwise forward conditional multivariate analysis was performed with icu characteristics, patient factors, and interventions found to be statistically significant in the univariate analyses . Analysis was confined to data from adult patients (> 16 years of age). Continuous variables were compared with the use of the student's t - test, analysis of variance, mann - whitney test or the kruskal - wallis test . Multivariate binary logistic regression analysis was performed to determine the independent predictors of icu mortality . A stepwise forward conditional multivariate analysis was performed with icu characteristics, patient factors, and interventions found to be statistically significant in the univariate analyses . Analysis was confined to data from adult patients (> 16 years of age). Continuous variables were compared with the use of the student's t - test, analysis of variance, mann - whitney test or the kruskal - wallis test . Multivariate binary logistic regression analysis was performed to determine the independent predictors of icu mortality . A stepwise forward conditional multivariate analysis was performed with icu characteristics, patient factors, and interventions found to be statistically significant in the univariate analyses . Data analysis was done for adult patients (n = 4038) from 120 icus . Details of participation are provided in the supplementary tables 14 . About 81 centers (67.5%) were considered adequately equipped, whereas 29 (32.5%) were categorized as not adequately equipped . Patients in medical icus had significantly higher severity of illness and mortality compared to patients in other types of icus . More than half the number of participating icus had a patient - nurse ratio of two or less patients per nurse . The primary apache iii diagnostic categories are summarized in table 3, and patient demographics, severity of illness, and outcomes are detailed in table 4 . Almost 546 patients died in the icu, and 183 patients were tds from the icu . The hospital mortality was 21.7% and included 694 patients who died in hospital and 183 tds . Median length of icu stay was 6 (interquartile range [iqr] 3 - 13) days, with significantly longer stays in nonsurvivors than in icu survivors [table 2]. Figures 1 and 2 show the distribution of apache ii scores and number of organ failures with the associated mortality . Almost 53.6% patients did not have any organ failure on the study day; mortality was 8.3% in these patients . Distribution of acute physiology and chronic health evaluation ii scores (number of patients in vertical bars on the y1 axis) and mortality rate (solid line on the y2 axis) distribution of organ failures (number of patients in vertical bars on the y1 axis) and mortality rate (solid line on the y2 axis) a subset of 1718 patients were admitted within 24 h of a study day, of which 1627 patients could have an apache ii diagnostic category assigned to enable calculation of the predicted mortality . Of these, 428 patients were predicted to die in hospital, whereas the observed hospital mortality was 286 . Apache ii over predicted mortality at scores above 19, as seen in figure 3 . Observed mortality (solid line) versus acute physiology and chronic health evaluation ii predicted mortality (dashed line) on the y2 axis in 1627 patients . Horizontal bars denote number of patients (y1 axis) mortality was higher in inadequately equipped icus compared to well - equipped icus (27.8% vs. 20.8%, p <0.001), despite similar apache ii scores . Medical admissions accounted for 77.1% of admissions; they had a higher severity of illness than surgical admissions and a significantly higher mortality . Similarly, mortality was significantly higher for admissions after emergency surgery than elective surgery [table 3]. Almost 124 patients were admitted after poisoning or drug overdose, including 72 organophosphorus or organochlorine poisoning, 5 snake bites and 21 unknown toxins . A sub - group of 1144 patients (28.3%) had svspss during the icu stay, with mortality of 34.0% . About 12.2% patients developed an infection during their icu stay and 1455 patients had a suspected or confirmed infection during the 24-h study period . Microbiological cultures and other tests for microbial identification were obtained in 2039 patients, and positive results were obtained in 35.9% . Overall, 1077 organisms were identified: 68.9% gram - negative organisms, 15.9% gram - positive organisms, 7.5% fungi, 2.4% mycobacteria, 1.7% viruses, and 1.1% malarial parasites . Various interventions in the icu are detailed in table 5 . On the study day, 5342 antibiotics were given to 72.4% patients, while 4.7% patients received antimalarials . About 60.7% of 2583 patients who did not have a suspected or confirmed infection on the study day received antibiotics, whereas 6.7% of the 1455 patients with a suspected or confirmed infection on the study day did not receive antibiotics . About 56.0% of medical patients and 72.3% of surgical patients received antibiotics without having a suspected or confirmed infection on that day . In patients receiving antimicrobials, patients receiving invasive mechanical ventilation (mv), vasopressors or inotropes (vis), and rrt had significantly higher mortality than those who did not (35.6% vs. 11.1%, p <0.001; 36.1% vs. 12.9%, p <0.001; and 31.5% vs. 16.2%, p <0.001, respectively). In 898 patients receiving vis, arterial and central venous catheters were inserted in 46.7% and 71.4% patients, respectively, and cardiac output and lactate levels were measured in 10.9% and 18.8% patients, respectively . Normal saline was the preferred fluid in more than 86% of the 1085 patients that received a fluid bolus . The results of the multivariate analysis of organizational and patient characteristics, severity of illness, and need for interventions are summarized in table 6 . The apache ii and sofa scores, public hospital icus, medical icus, inadequately equipped icus, medical admission, self - paying patient, the presence of svspss shock, acute respiratory failure (pao2 /fio2 ratio <300) or cancer, and the need for a fluid bolus and mv were independent predictors of mortality . Patients had moderate severity of illness, as evidenced by an apache ii and sofa scores of 17.4 9.2 and 3.8 3.6, respectively . This widespread practice of tds is contrary to that reported in one single center study and one study of end - of - life care practices in four hospitals in mumbai . It suggests that end - of - life care in indian icus may be suboptimal and is probably related to the unresolved legal status of withholding and withdrawal of life - sustaining treatments in critically ill patients in india . It may also perhaps reflect a small proportion of cases in which the patients or families wishes to have a peaceful death at home were respected . We assumed that all tds from the icu eventually died and included these in the icu mortality . Admittedly, while a small proportion of tds may have survived, merely counting the number of patients who died in icu would underestimate the number of nonsurvivors . The low smr observed in a subset of patients in this study must be interpreted with caution . It may reflect the quality of care, but could also be due the visibly poor calibration of apache ii . Variables such as the glasgow coma scale may have been inaccurately recorded in sedated patients, with resultant erroneously high apache ii scores . Our mortality in patients with apache ii scores above 19 and in patients with 3 or more organ failures was lower than that predicted by apache ii or that observed by vincent et al . In their study of the sofa score . A formal evaluation of apache ii as well as other scoring systems is necessary to determine the scoring system that works best in our circumstances . Our overall icu mortality rate of 18.1% was higher than the 16.2% rate observed in the intensive care over nations (icon) audit . Our icu mortality of 34.0% from svspss was similar to the 36.7% mortality in a multicenter study of 150 icus from 16 asian countries . However, it was higher than that reported in the impress study (28.4% globally and 30.8% for asia) and in the icon study (258%). In mechanically ventilated patients, mortality of 33.8% in our study was more than the 28% mortality reported in the third international survey on mv . Thus, improvements are required in the organization and delivery of critical care in indian icus . It is essential to increase health insurance plans and government schemes to reduce the economic burden on individuals and families . Absence of adequate facilities in the hospital may lead to inadequate or delayed care or may necessitate transport that may increase the risk of complications, with consequent increase in mortality . A study by parikh and karnad in a large public hospital icu in mumbai had an observed mortality of 36%, smr of 1.67 and lower intensity of interventions . This could represent either variations in case mix and clinical practice or could reflect inadequate facilities as well as patients inability to afford expensive interventions . Perhaps, greater spending to attain higher levels of care in terms of equipment, drugs, interventions, staffing, and organization may lead to better outcomes with greater cost - effectiveness . A more detailed analysis that includes costs and the participation of more public hospital icus is essential to answer these questions . The presence of a training program in critical care medicine was not associated with improved outcome on multivariate analysis . A majority of the programs were of 1-year duration, and there have been variations in the intensity and quality of training between the accredited icus . The content and structure of the program, as well as the accreditation process, may need to be reviewed . We defined a closed icu as one where the final orders were written by the intensive care team . This includes the completely closed model, the semi - closed, transitional, or mandatory consult model or the high - intensity multidisciplinary care models . Studies of the impact of high - intensity models on outcome have shown mixed results, with several studies showing improved mortality and better resource utilization, some showing no effect and one showing higher mortality . However, we were unable to demonstrate that closed units were associated with lower mortality, and this study was not designed to detect other benefits of a closed icu model . This study confirms that gram - negative infections are predominant in india (69%), as opposed to gram - positive infections in western countries . While antibiotics were given in 60.7% of noninfected patients, some patients may have been completing their course of antibiotics, and a proportion of noninfected surgical patients may have received antibiotics as perioperative prophylaxis . Nevertheless, these data suggest that infection control practices and antibiotic stewardship need to be strengthened in indian icus . In patients receiving vis, insertion of arterial catheters, cardiac output monitoring and estimation of lactate levels were infrequently performed, perhaps because monitoring was not either available or was not utilized . Arguably better - performing icus that were motivated and willing to share data contributed to the study, while icus with poor performance did not . Only 11% of icus and 10% patients were from the public sector, but this may represent the general distribution of icus between the private and public sectors in india . Source data verification was not performed . We assumed that the investigators correctly applied the definitions and diagnostic criteria for sepsis and other conditions such as malaria, dengue fever, etc ., some data were incomplete, especially for the length of icu stay and hospital stay . However, there was no systematic exclusion of data, and the information obtained would still remain valuable . We were unable to obtain data on physician staffing patterns, protocols and night - time coverage by intensivists . The strengths of this study include a large number of icus and patients from all regions of the country and from different types of icus . Data from this study can be used to determine the prevalence of different conditions in indian icus, as well as to determine the baseline mortality rates in important subgroups of patients, e.g., 34.0% in patients with svspss, 33.8% and 36.1% in patients requiring mv and vis, respectively . We did not estimate the smr from the apache ii score for all patients as we calculated the score on the study day, and not in the first 24 h of icu admission . However, we did estimate the smr for those patients whose apache scores were calculated within 24 h of icu admission . We only analyzed data from adult patients as there were only 171 children in the database . This multicenter, point - prevalence study of 4038 adult patients from 120 icus is a snapshot of intensive care in india . Highlights include a moderate severity of illness with relatively high mortality in patients with severe sepsis and septic shock, patients on vasopressors or inotropes or receiving mechanical ventilation . Antibiotics are often given in noninfected patients, monitoring practices in seriously ill patients can be improved . Self - paying patients, public hospitals icus, and inadequately equipped icus are independently associated with a worse outcome . Terminal discharge from the icu is widely practiced and legal, social, and other issues related to end - of - life care need to be resolved.
Porcelain - fused - to - metal crowns have been used as predictable materials since the 1960s, owing to their mechanical strength and low cost.12 however, porcelain veneer failure has been reported as the major cause for the replacement of metal - ceramic restorations.34 the fracture of veneering porcelain may result from trauma, improper metal framework design, incompatibility between the thermal expansion coefficient of the porcelain and core, inadequate tooth preparation, inadequate occlusal adjustment, and intraceramic defects.56 the rate of failure caused by porcelain fracture is reported in the literature to be approximately 2 - 16%,4789 and the majority of failures (65%) are observed in the anterior region, and mainly in the maxilla (75%), predominantly at the labial surface.10 also, failure may often occur in the anterior regions, presenting a serious aesthetic problem . The immediate replacement of failed complex prostheses is often impossible though, as it requires additional time, effort, and expense . In this situation, repair is a suitable method to rehabilitate the contour and color of fractured restorations . Intraoral repair using a bis - gma composite light - cured resin can be an alternative method that offers great benefits due to its superior aesthetics, color stability, and ease of application.11 various techniques for the preparation of exposed surfaces have also been introduced to improve the bonding qualities between metal or porcelain substrates and resin composites . Tribochemical silica coating is an effective surface treatment regardless of whether the fracture takes place in metal or porcelain, or is a combined exposure.1121314 hydrofluoric acid etching followed by the application of a silane coupling agent also promotes good results in the preparation of a ceramic surface, and it is commonly used in clinics due to its simplicity.15161718 before the application of hydrofluoric acid, the exposed surface can be roughened by a diamond bur or by sandblasting with aluminum oxide, both of which can affect the bond strength . Several researchers reported that mechanical roughening by both a diamond bur and sandblaster was effective for porcelain repair.1920 the best results were achieved with a bur by leibrock and colleagues3 and with sandblasting by tulunoglu and beydemir.21 in many studies, however, the comparison of bonding qualities with different roughening methods was performed using different resin composite repair systems . For this reason, a comparison based upon the use of the same repair system is needed in order to confirm the differences in roughening procedures . The purpose of this study was to evaluate the bond strength of two porcelain repair systems for the metal and porcelain surfaces: the repair system i (intraoral repair kit, bisco inc ., schaumburg, il, usa) and the repair system ii (cojet intraoral repair system, 3 m espe ag, seefeld, germany), and the differences in bond strength obtained by two different surface treatments with identical repair system: 1) roughening with diamond bur and 2) airborne - particle abrasion . The null hypothesis tested was that, regardless of the repair systems and surface treatments, the bond strength between the core surface (metal and porcelain) and resin composite veneer would not be different among the groups . All the components of the resin composite repair systems and their chemical compositions used in this study were listed in table 1 . Fifteen disks (1.0 mm thickness 8.0 mm diameter) were fabricated with feldspathic porcelain (vintage mp, shofu inc ., another fifteen specimens were made of a nickel - chromium alloy (bellabond plus, bego, bremen, germany) with identical dimensions with porcelain disks . All disks were then placed into the stainless steel molds and fixed with an auto - polymerizing poly - methyl methacrylate resin (ortho - jet, lang, wheeling, il, usa). After polymerization, the resin cylinders were separated from the molds, and the test surfaces were smoothened with 220-grit abrasive paper and carefully polished (sof - lex extra thin, 3 m espe ag, seefeld, germany). The specimens, either feldspathic porcelain (p) or nickelchromium metal alloy substrates (m), were divided into three groups according to the applied repair systems and surface treatments: 1) application of repair system i (intraoral repair kit, bisco inc ., schaumburg, il, usa) with surface roughening with diamond bur (group dp and dm), 2) application of repair system i with airborne - particle abrasion (group sp and sm), and 3) application of repair system ii (cojet intraoral repair system, 3 m espe ag, seefeld, germany) (group cp and cm). For the group dp, the porcelain surfaces were roughened with a diamond bur and 4% hydrofluoric acid gel (porcelain etchant, bisco inc ., schaumburg, il, usa) was applied for 5 minutes . A silane coupling agent (porcelain primer, bisco inc ., schaumburg, il, usa) was applied onto the each treated surface, which was allowed to react for 30 seconds, and air - dried ., schaumburg, il, usa) was subsequently applied, air - thinned and photo - polymerized for 10 seconds with a visible light curing unit (elipar freelight2, 3 m espe ag, seefeld, germany). After a glass matrix with a 6.0 mm diameter was placed on the specimens in order to obtain an equal bonding area, a hybrid resin composite (aelite all - purpose body, bisco inc ., schaumburg, il, usa) was applied with a thickness of approximately 1.5 mm, and cured for 20 seconds . After the matrix was removed, a metal primer (z - prime plus) was brushed and airthinned for 5 seconds according to the manufacturer's instruction . An adhesive (one - step plus) was then applied, air - thinned and photo - polymerized for 10 seconds with a visible light curing unit (elipar freelight2). The mixture of the catalyst and base (opaquer catalyst and opaquer base universal, bisco inc ., schaumburg, il, usa) was applied to the prepared surface and photo - polymerized for 10 seconds . A hybrid resin composite (aelite all - purpose body) was applied in the same method as previous procedures with the porcelain substrates . For the group sp and sm, the test surfaces were subjected to airborne - particle abrasion (airsonic mini sandblaster, hager & werken, duisburg, germany) with 50-m aluminum oxide particles (cobra, renfert, hilzingen, germany) from a distance of 10 mm at a pressure of 0.3 mpa . The subsequent procedures for the application of porcelain repair system on porcelain and metal disks were identical as those used in the group dp and dm, respectively . For the group cp, 30-m silica - coated alumina particles (cojet sand, 3 m espe ag, seefeld, germany) were blasted from a distance of 10 mm at a pressure of 0.3 mpa . After blasting, the porcelain substrates were coated with -mps silane (espe sil, 3 m espe ag, seefeld, germany) and then air - dried . For the group cm, a primer (z - prime plus) was applied and dried for 5 seconds on the metal substrate . A mixture of sinfony opaque and catalyst (3 m espe ag, seefeld, germany) was subsequently applied and photo - polymerized for 10 seconds according to the manufacturer's instruction . For both core specimens, an adhesive (adper single bond 2 adhesive, 3 m espe ag, seefeld, germany) was applied, air - thinned and photo - polymerized for 10 seconds . The resin composite (filtek z250, 3 m espe ag, seefeld, germany) was layered on the test surface of each substrate with a thickness of approximately 1.5 mm using customized matrix and cured using the same protocol as with the group dp . All specimens were then stored in 37 distilled water for 15 hours and thermocycled between 5 and 55 for 1000 cycles with a 30 seconds - dwell time . After cycling, they were additionally stored in 37 distilled water for 192 hours before being subjected to a shear load ., norwood, il, usa) with a 10-kn load cell and a 0.5-mm / min crosshead speed, a flat - end apparatus was used to direct parallel shearing forces as close as possible to the resin/ substrate interface (fig . The shear load in newtons at the point of failure was noted, and force was calculated in mpa . The mode of failure was recorded as being adhesive (failure at the substrate - resin interface), cohesive (failure within the substrate), and mixed (adhesive and cohesive). All data (mpa) were analyzed using a statistical software package (pasw statistics 18, ibm spss, chicago, il, usa) that employs the kruskal - wallis analysis and the bonferroni post - hoc test for the multiple comparison between the groups with identical core surfaces to determine the effect of repair system on the adhesion to the resin composites . Additional statistical analyses for the influence of the substrates on the resin bonding qualities according to various repair methods were performed with mann - whitney test . The mean shear bond strengths of porcelain and metal specimens were listed in table 2 . On the porcelain surface, the application of airborne - particle abrasion with repair system i (intraoral repair kit) significantly improved the core - resin composite bonding compared to other repair methods (p <.05). The repair system i applied with diamond bur roughening and the repair system ii (cojet intraoral repair system) were not significantly different in core - veneer bond strength . On the metal surface, application of the repair system ii was significantly beneficial to the core - resin composite bonding than other methods using repair system i (p <.05). Regardless of the surface roughening methods, the repair system i showed significantly higher bond strength of resin composite to the porcelain substrate than to the metal core (p <.05). On the contrary, the repair system ii showed similar bonding quality for both metal and porcelain core to the resin composite (p>.05). Values followed by identical uppercase letters present no statistical differences between groups with identical core materials . Lowercase letters with the same subscript show no significant differences between groups with identical repair methods . Dp and dm group: porcelain and metal specimens treated with repair system i (intraoral repair kit) after diamond bur roughening, respectively; sp and sm group: porcelain and metal specimens treated with repair system i (intraoral repair kit) after airborne - particle abrasion; cp and cm group: porcelain and metal specimens treated with repair system ii (cojet intraoal repair system). In the porcelain specimens, mixed type of failure was present 100% in the dp group, 80% in the sp group, and 60% in the cp group . In the metal specimens, adhesive failure occurred when the repair system i was applied after diamond bur roughening . In this study, one repair system with tribochemical silicacoating (cojet intraoral repair system) was compared with another repair system (intraoral repair kit) with two different surface roughening treatments recommended by the manufacturer . Based on the results, the null hypothesis was rejected since the bond strength of substrate to resin composite was significantly different among the groups with different repair methods . It could be inferred that using the intraoral repair kit with airborne - particle abrasion was helpful in repairing fractured porcelain surface, while cojet repair system was beneficial to restore exposed metal surface with resin composite . Each group of metal specimens displayed a unique failure mode in response to different repair methods . The metal specimens treated by the repair system i (intraoral repair kit) after diamond bur roughening displayed adhesive failure, and also showed low bond strength . In the specimens roughened by sandblasting, the opaque porcelain was often partially detached . Also, a greater part of the opaque porcelain failed in metal specimens treated by the repair system ii (cojet repair system) than failed using the repair system i. the failure mode of the metal specimens tends to reflect the core - resin bond strength values, which demonstrated a stronger bond between the exposed metal surface and resin composite in the groups with repair system ii than in the groups with repair system i. various techniques such as acid etching, sandblasting, silanization, and application of metal primer have been introduced for the repair of fractured metal - ceramic restorations . Acid etching of feldspathic porcelain creates micromechanical undercuts that have a decisive effect for better adhesion.222 many studies have reported that a combination of micromechanical roughness and silane application to the porcelain creates durable bonding.15161718 silane is a dual functional monomer that consists of a silanol group that reacts with the ceramic surface, and it contains a methacrylate group that co - polymerizes with the resin matrix of the composite.2 silane coupling agents are known to enhance the wettability of glass substrates by resin composites and are also known to increase the mechanical and chemical bonding of resin composite to ceramics.23 the alloy primer or composite containing diphosphate monomer (mdp), which has a phosphate ester group to bond directly to metal oxides, has been assessed as having superior bonding durability to base metal alloy.2425 in addition, a tribochemical silica coating was also investigated that could provide increased bond strength even to metal, and with less influence upon the surface materials.1121314 to the contrary of our findings, ozcan and coworkers26 reported that the' intraoral repair kit' revealed significantly lower bond strength values than those of the' cojet repair system' for surface of feldspathic porcelain after thermocycling, which were also tested in this study . Surface roughening with diamond bur or particle abrasion before using the porcelain repair system, omitted in the previous study, could be the reason of the difference between the results of the studies.27 the application of' intraoral repair kit' with sandblasting seemed to be an effective pretreatment that improves the porcelain - to - composite bond . It has been reported that microscopic cracks were produced when alumina particles were sandblasted to a ceramic surface, and that hydrofluoric acid penetrated along the groove and resin was able to penetrate deeply into the micromechanical undercuts.15 adhesive materials containing 4-meta (4-methacryloxyethyl trimellitate anhydride) as well as mdp have been shown to have good bonding properties to base metal alloys.112528 improved bond strength to a metal surface might thus be expected if repair materials containing 4-meta or mdp were used . A prehydrolyzed, 3-methacryloxypropyl trimethoxysilane coupling agent (-mps) was then applied that forms covalent bonds between the silica particles and the adhesive.29 because alumina particles coated with silica were bound to the metal surface mechanically, the cojet repair system might display a better bond strength to alloy than the intraoral repair kit . The resin bond strength of the cojet repair system was reported to have a range of 14.5 - 22.4 mpa to porcelain and 15.95 - 25.24 mpa to metal.1121326 one study1 reported that the cojet repair system displayed significantly higher bond strength to nickel - chromium alloy than to porcelain . In addition, haselton and coworkers12 reported no significant difference between the bond strength of a high noble alloy and feldspathic porcelain treated by the cojet repair system . Ozcan and niedermeier10 investigated 289 metal - ceramic restorations that were fractured during mean 34.6 months . The overall cumulative survival rate was 89%, according to the kaplan - meier curve method . A repair procedure thus seems to be an alternative treatment, and moisture control is essential for a successful result . The specimens in this study were thermocycled for 1,000 cycles between 5 and 55, as in previous studies.11314 after a review of the literature on thermal cycling procedures, gale and darvell30 proposed that some 10,000 cycles might represent one service year, based on 20 to 50 cycles being equivalent to a single day . Thermal stress would weaken the porcelain - composite resin bond because of differences in the coefficients of thermal expansion and bond deterioration via hydrolysis . Further studies subjecting a greater number of specimens to more thermal cycles and to a long period of storage in water should be explored in order to provide more reliable information about the repair systems studied . Within the limitations of this study, sandblasting and application of the repair system i (intraoral repair kit) can be suggested in the case of a fracture with exposed area of body porcelain . If the fracture extends to the metal surface, the repair system ii (cojet repair system) is worthy of consideration.
In developing countries, cervical cancer is the commonest malignancy among women, and india is no exception . In our hospital, it represents about 24% of the overall malignancies in females . Due to lack of adequate screening program combined with very low health consciousness and infrastructural deficiencies, majority of these patients present at an advanced stage, mostly in iiib and some in iib as per figo staging system . Treatment of choice in these patients is radiotherapy, which is a combination of both external beam radiotherapy (ebrt) and intracavitary brachytherapy (icrt) of which former one is usually combined with concurrent chemotherapy . Aim of ebrt is to control the pelvic disease as well as to reduce the size of central tumor, later of which is ultimately controlled by icrt . Brachytherapy sources have undergone a full evolution from manual pre - loaded low - dose - rate (ldr) to remote controlled after loaded high - dose - rate (hdr). Advantages of hdr sources are manifold, e.g. Elimination of radiation exposure to the attending physician and other health personnel, patient convenience, better dose optimization due to short overall treatment time, greater sparing of the rectum and bladder by temporary spacing, inter - digitations with ebrt, out - patient basis treatment delivery resulting in decreased need of hospitalization etc . . In addition, most modern afterloaded sources are stepping source that allow optimization of dose distribution by varying the dwell time and positions . The term events occurring within 30 days from the date of insertion of brachytherapy necessitating hospitalization or death. Due to few number of cervical cancer patients in developed countries and still fewer number in advanced stage, information in this respect in world literature is sparse . Here, we are reporting our experience of acute complications following hdr brachytherapy in gynecological malignancies, and have tried to identify the causative factors to further reduce the already low rate of acute complications . From february 2004 to december 2012, a total of 1947 patients with gynecological malignancies were treated by ebrt and icrt . Most of them were diagnosed with cervical cancer, a minority with endometrial cancer . Before starting treatment, patients were evaluated thoroughly and the evaluation criteria were: histologically proved carcinoma of the cervix or endometrium and completed ebrt;staging as per figo recommendation;hematological parameters were as follows: a) hemoglobin: 10 gm%; b) platelet count: 100 000/mm; c) serum creatinine: 1.5 times of normal value; d) bleeding and clotting times: within normal limits;electrocardiogram: within normal limits;post - prandial blood sugar: within normal limits;x - ray chest and pulmonary function tests: within normal limits;ultrasonography / ct scan of whole abdomen, particularly to note status of pre and para aortic lymph nodes;mri of pelvis, if possible;informed consent . Histologically proved carcinoma of the cervix or endometrium and completed ebrt; staging as per figo recommendation; hematological parameters were as follows: a) hemoglobin: 10 gm%; b) platelet count: 100 000/mm; c) serum creatinine: 1.5 times of normal value; d) bleeding and clotting times: within normal limits; electrocardiogram: within normal limits; post - prandial blood sugar: within normal limits; x - ray chest and pulmonary function tests: within normal limits; ultrasonography / ct scan of whole abdomen, particularly to note status of pre and para aortic lymph nodes; mri of pelvis, if possible; patients, who did not fulfill the above mentioned criteria 3 - 6, were first given appropriate management and then treatment for malignancy began . Pre - treatment patient characteristics (n = 1947) all patients had already been treated by ebrt at a dose of 50 gy/25 fractions/5 weeks by parallel opposed antero - posterior beams to the whole pelvis in cobalt-60 machine (theratron 780e, theratronics, canada). Bulky patients having inter - field distance 18 cm, were treated by four field box technique . Most of the cervical cancer patients had also received concurrent chemotherapy with cisplatin 40 mg / m weekly 5 such . For cervical cancer patients, an icrt dose of 7 gy at manchester point a weekly for 3 insertions was recommended . In patients with intact uterus, an uterine tandem and a pair of an ovoids in the vaginal fornices rectal separator followed by tight vaginal gauze packing was done to increase the distance between the radiotherapy sources and anterior rectal and posterior bladder walls . Post operative endometrial cancer patients received an icrt dose of 6 gy at a depth of 5 mm from the surface of the vaginal mucous membrane for 3 fractions at one week apart by vaginal cylinder . All the icrt procedures were carried out on outdoor patient department (opd) basis under deep sedation . A foley's catheter was inserted into the urinary bladder and radio - opaque solution was pushed into the bulb of the catheter to delineate the posterior bladder wall . Because of the narrow width of the intrauterine tandem, cervical dilatation was rarely required . In some patients where intrauterine tandem could not be administered because of anatomical distortion or cervical fibrosis due to disease process, only ovoids had to be used . After completion of the insertion process and vaginal packing, a rubber tube containing thick barium sulphate solution was inserted into the rectal canal, and treatment planning was done with the help of c - arm visualization (mexicon 2000, m e x - ray india pvt . Ltd, india) and treatment planning system (tps, nucletron, an elekta company, elekta ab, stockholm, sweden) till 2010 . Since 2010, c - arm was replaced by dedicated computerized tomography (ct scan, somatom emotion 16, siemens ltd, kolkata, india). When there was a sudden loss of resistance at the time of uterine sound / tandem insertion or uterine cavity length was found to be unusually long, an uterine perforation was suspected, to be confirmed or excluded by immediate pelvic usg / ct scan, and either the procedure was postponed or continued . All patients with uterine perforation and extensive vault / vaginal lacerations were immediately hospitalized and managed accordingly . When postponed, the icrt procedure was resumed one week after the patient was stabilized . When perforation either complete or partial has occurred in any attempt, post insertion usg / ct scan was done routinely in subsequent insertions even when perforation was not suspected . Prophylactic antibiotic was prescribed in all patients who had intrauterine manipulation or vaginal laceration and oral analgesics to all patients . After completion of treatment, patients were followed up at two weeks interval for two months and then at six weeks interval for the next six months . Non compliance to follow up was almost 3% at one month, which gradually increased to 11% at six months . After that period, follow up interval was gradually increased and follow up continued till death with late complications documented, which will be discussed at a later date . All patients had already been treated by ebrt at a dose of 50 gy/25 fractions/5 weeks by parallel opposed antero - posterior beams to the whole pelvis in cobalt-60 machine (theratron 780e, theratronics, canada). Bulky patients having inter - field distance 18 cm, were treated by four field box technique . Most of the cervical cancer patients had also received concurrent chemotherapy with cisplatin 40 mg / m weekly 5 such . For cervical cancer patients, an icrt dose of 7 gy at manchester point a weekly for 3 insertions was recommended . In patients with intact uterus, an uterine tandem and a pair of an ovoids in the vaginal fornices rectal separator followed by tight vaginal gauze packing was done to increase the distance between the radiotherapy sources and anterior rectal and posterior bladder walls . Post operative endometrial cancer patients received an icrt dose of 6 gy at a depth of 5 mm from the surface of the vaginal mucous membrane for 3 fractions at one week apart by vaginal cylinder . All the icrt procedures were carried out on outdoor patient department (opd) basis under deep sedation . A foley's catheter was inserted into the urinary bladder and radio - opaque solution was pushed into the bulb of the catheter to delineate the posterior bladder wall . Because of the narrow width of the intrauterine tandem, cervical dilatation was rarely required . In some patients where intrauterine tandem could not be administered because of anatomical distortion or cervical fibrosis due to disease process, only ovoids had to be used . After completion of the insertion process and vaginal packing, a rubber tube containing thick barium sulphate solution was inserted into the rectal canal, and treatment planning was done with the help of c - arm visualization (mexicon 2000, m e x - ray india pvt . Ltd, india) and treatment planning system (tps, nucletron, an elekta company, elekta ab, stockholm, sweden) till 2010 . Since 2010, c - arm was replaced by dedicated computerized tomography (ct scan, somatom emotion 16, siemens ltd, kolkata, india). When there was a sudden loss of resistance at the time of uterine sound / tandem insertion or uterine cavity length was found to be unusually long, an uterine perforation was suspected, to be confirmed or excluded by immediate pelvic usg / ct scan, and either the procedure was postponed or continued . All patients with uterine perforation and extensive vault / vaginal lacerations were immediately hospitalized and managed accordingly . When postponed, the icrt procedure was resumed one week after the patient was stabilized . When perforation either complete or partial has occurred in any attempt, post insertion usg / ct scan prophylactic antibiotic was prescribed in all patients who had intrauterine manipulation or vaginal laceration and oral analgesics to all patients . After completion of treatment, patients were followed up at two weeks interval for two months and then at six weeks interval for the next six months . Non compliance to follow up was almost 3% at one month, which gradually increased to 11% at six months . After that period, follow up interval was gradually increased and follow up continued till death with late complications documented, which will be discussed at a later date . Cervical cancer patients had a median age of 41 years (32 - 69 years) and only 12% of them were postoperative . Endometrial cancer patients had a median age of 58 years (45 - 74) and 79% of them were postoperative . Regarding staging, stage iii was the most common (62%) in cervical cancer patients followed by stage ii (28%), whereas, most of the endometrial cancer patients were of stage i (41%) and ii (38%). Most of the patients of both the groups had karnofsky performance status 90 . Pre - icrt evaluation results have also been shown in table 1 . In comparison to endometrial cancer, more cervical cancer patients had hemoglobin level <10 gm% (22% vs. 11%) and deranged kidney function (17% vs. 10%). These patients received blood transfusion and management from the nephrologists before starting icrt . Similarly, abnormal electrocardiogram and raised blood sugar were more common in endometrial cancer patients (16% vs. 0.8% and 23% vs. 0.6%), and were treated accordingly . Though three icrt insertions were planned in all patients, the prescribed treatment could not be completed in all of them . Treatment with both tandem and ovoids was attempted in 1468 cervical and 59 endometrial (with intact uterus) cancer patients . Tandem could not be inserted in 54 (3.67%) cervical and 7 (11.86%) endometrial cancer patients . One of them died due to acute complications, following complete uterine perforation, and the rest did not turn up for second insertion for unknown reason . Thirteen of them had complete uterine perforation of whom 4 died, rest recovered, 16 partial uterine perforation, vault laceration in 2, deep vein thrombosis in 2, fever in 2, and the rest did not turn up for unknown reason . In endometrial cancer patients, 4 of them completed only two insertions; 1 of them died following complete perforation and the other 3 did not turn up 1 of whom had deep vein thrombosis . Eleven cervical and 3 endometrial cancer patients detected with complete uterine perforation after 3 insertion of whom, 5 cervical and 1 endometrial cancer patient died, rest recovered . Similarly, partial uterine perforation was detected in 29 cervical and 3 endometrial cancer patients after 3 insertion and all recovered . Only tandem and ovoids patients the median insertion time was about 35 minutes in our patients . Once the tandem and ovoids were in position, rectal separator applied and vaginal packing done, the patients were placed in supine position during the imaging, calculation, and irradiation procedure . Another 40 - 50 minutes were required for imaging and calculation followed by 8 - 35 minutes for dose delivery depending upon the source output . When uterine perforation occurred in any insertion attempt, routine post insertion usg / ct were done in subsequent attempts, even when perforation was not suspected . In our institution, in the above mentioned 9 years period, only 239 acute events had occurred consisting of 12 mortalities and 227 morbidities . Thirteen percent (2.13%) patients were detected to have complete uterine perforation by usg and the figure rose to 4.12% in post - ct era . But if we consider the total number of insertions done, then the percentages become 0.73% and 1.38% in pre- and post - ct era . These percentages were calculated based on the number of patients in whom tandem insertion was attempted . During this period, 12 patients died due to uterine perforation, 11 in the pre - ct, and 1 in the post - ct scan era . In the pre - ct period, usg were done only in the suspected cases of perforation, and in 10 of the patients perforation was either not suspected or not detected . They came back after 3 - 4 days with features of peritonitis; perforation detected retrospectively and could not be salvaged . One patient in the pre - ct era and 1 who died in the post - ct era, perforation was detected during tandem insertion, admitted in the hospital, treated conservatively, but the gut injury remained undetected and died of peritonitis . Rest 25 patients, in whom complete uterine perforation was detected during icrt procedure, were kept admitted, treated conservatively, and recovered . Most of the partial uterine perforations were detected in the post - ct era and were managed conservatively with antibiotics, fluid, and blood transfusion, when needed . Vault or vaginal lacerations were treated with tight gauge packing under anesthesia, sedation, antibiotics, and blood transfusion, if required, and only 2 of them required suturing . Description of acute events (n = 239). Only in tandem and ovoids patients; total 4285 insertions cm conservative management, bt apart from scientific reasons, cost effectiveness is a compelling criterion of its popularity . Due to vast number of patients suffering from gynecological malignancies, particularly from carcinoma cervix of advanced stage combined with relatively few number of available brachytherapy units, time has come when we will be cautious about the acute and late effects of hdr brachytherapy . It is dangerous to assume that hdr brachytherapy is associated with less acute complications than ldr brachytherapy . As patients are treated on opd basis, they can't be observed for a certain length of time post icrt and acute complications may progress to a certain extent before diagnosis, leading to increased morbidity and mortality . Acute complications related to ldr brachytherapy are gynecological malignancies and have been described in some literature . Analyzed acute complications in 143 insertions (100 patients) of tandem / colpostat in cervical cancer patients in 20 of whom insertions were done under ultrasound guidance . These included uterine perforations (4), vaginal lacerations (2), and 1 instance of bladder perforation . Postoperative complications (e.g., fever, bowel obstruction, exacerbation of chronic obstructive pulmonary disease, cardiac complication) occurred in 54 out of 143 placements . A multivariate analysis showed that underlying copd predisposed to postoperative complications during the first implant, and that age over 60 years independently predicted for complications during any implant . They concluded that intraoperative complications are rare and use of ultrasonography allows safe tandem insertion . Incidence of uterine perforation was 2.79% in corn's report, and 2.47% of insertions in our series . Evaluated the incidence and severity of perioperative morbidity with standard tandem and ovoid insertions in gynecological malignancies . Five percent of the insertions had to be removed emergently secondary to presumed sepsis, exacerbation of chronic obstructive pulmonary disease, hypotension, change in mental status, and myocardial infarction / congestive heart failure . Sarkaria et al . Assessed the 30-day morbidity and mortality rates for patients with an intact uterus undergoing hdr brachytherapy, and the overall acute events occurred in 9.5% of patients . Their overall morbidity and mortality rates were similar to ldr brachytherapy, but higher than other hdr reports and age, turned out to be the most significant predictive factor . The overall acute events occurred in 5.57% of our insertions, which is much lower than reported by sarkaria et al . . Reported 30 days morbidity and mortality rate of 6.4% and 1.5%, respectively, following hdr brachytherapy . Reported 4.2% and 2.1% of morbidity and mortality rates, respectively, in gynecological malignancies, and his series involved inoperable endometrial carcinoma patients . Our morbidity rate was a bit higher (5.29%), but mortality rate (0.28%) was much lower than chao et al . However, the largest trial to date has been published by knocke et al ., in which 280 medically inoperable endometrial cancer patients were treated by hdr brachytherapy and experienced no acute complication . Petereit et al . Reported 30 day morbidity and mortality for cervical and medically inoperable endometrial carcinoma patients as 5.5% and 1.6%, and 9.8% and 7.3%, respectively, with hdr brachytherapy . They identified certain risk factors associated with acute complications e.g. Age by decades, performance status, cardiac problems, american society of anesthesiologists (asa) physical class system, positive medical history etc . A relative risk of 2.0 per decade implies that an 80 year old woman has an estimated 16 fold higher risk than a year old women . Have also stressed on age, cardiac problems, type of anesthesia used etc, as factors influencing acute complications . In our patients, rate of acute complications were significantly low and we have tried to analyze the factors responsible for this low rate of acute complications in comparison to published reports . As age has turned out to be the most important factor influencing acute complication, it is quite clear why acute complications were less in our patients . Median age of our cervical and endometrial cancer patients was 41 years (range: 32 - 69 years) and 58 years (range: 45 - 74 years), respectively, whereas the mean age of petereit's patients was 50 years (range: 23 - 88 years) and 63 years (range: 37 - 81 years), respectively . Petereit et al . Reported an incidence of 7.3% and 1.8% respectively, in endometrial cancer and overall patients . But, incidences of deep vein thrombosis were 1.2% and 1.0%, respectively, in dusenbery and corn series [2, 5]. Similarly, we have observed only 16 (0.37%) incidence of clinically significant deep vein thrombosis in our patients . The cause of this low incidence of deep vein thrombosis probably lies in the racial variation, age, procedural time, and pre - treatment management . The median procedural times in petereit series were 133 minutes and 160 minutes, respectively, for cervical and endometrial cancer patients, and the patients were kept in dorsal lithotomy position during this whole period of procedure . This low procedural time is probably responsible for the low incidence of deep vein thrombosis in our patients . Accurate positioning of the intra - cavitary applicators are critical for delivering appropriate doses of irradiation to the target volume while keeping the doses to the surrounding organs at risk below their tolerance limits . Consequently, unrecognized uterine perforation may lead to under dosage of the target volume, compromising local control probability . In addition, the perforating applicator may come in direct contact or in vicinity of the organs at risk, leading to their exposure to excessive doses, resulting in acute and long - term gastrointestinal, and genitourinary complications . Incidence of uterine perforation, particularly partial perforation during tandem insertion is probably underappreciated in our series . Complete perforation was 0.73% in pre - ct scan era, but 1.38% in post - ct scan period . As usg were done only in suspected cases of perforation, it may easily be assumed that a lot of sub - clinical perforations have been missed . Similarly, incidence of partial perforation rose from 0.38% to 6.46% with the introduction of routine ct scan . Petereit et al . Documented a 2% incidence of uterine perforation in their patients, but many of these perforations were partial, dissected the anterior or posterior uterine wall rather than puncturing the uterine serosa . . Tried to determine the incidence of uterine perforation by 3d imaging with the applicators in position, and found the incidence to be 3% only and all cases were managed conservatively . Uterine perforation occurs more frequently in patients with anatomical distortions of the cervix and/or cervical stenosis due to advanced disease, post irradiation fibrosis and previous cone biopsy . Patients over 60 years are particularly predisposed to uterine perforation, probably due to vaginal atrophy and anatomical distortion of the cervix . Other predisposing factors include retro - verted / retro - flexed or extremely ante - verted / ante - flexed uterus . In our institution, icrt was started one week after completion of ebrt and therefore chances of anatomical distortion of cervix or post radiation fibrosis were much less and cone biopsy was not done in any of them . Conscious sedation the goal of which was to relieve anxiety and pain and to promote amnesia . Petereit series had observed almost 10% mortality in the inoperable corpus group . As we have used deep sedation (combining inj . Diazepam and pentazocin) in our patients, no mortality in this respect has been noticed . High - dose - rate brachytherapy is gaining popularity in developing countries where it is mostly needed as almost 30% of malignancies of women of these countries are gynecological . But probably none has published their reports of acute complications of hdr brachytherapy in gynecological malignancies, particularly in opd settings . Our study showed that the acute complications of intracavitary brachytherapy can be minimized by pre - treatment management of associated comorbidities, decreasing the duration of operative lithotomy position, routine use of 3d imaging in all insertions etc.
There are two clinically recognized distinct variants of vitiligo based on distribution of depigmented areas; generalized and localized (1,2). Generalized or bilateral symmetrical form of vitiligo is a disease that destroys skin and mucosal membranes melanocytes progressively, and in some cases could involve ears and eyes (3,4). Segmental vitiligo is an uncommon form of localized vitiligo, characterized by dermatomal distribution . It is often unilateral and asymmetrical that never crosses the midline of body (1,4,5). In this form of the disease, it is believed that in the vast majority of the patients with segmental vitiligo melanocytes of the hair follicles are as well affected, resulting in leukotrichia (6). The most commonly used medical therapies for localized vitiligo include topical steroids and calcineurin inhibitors such as tacrolimus (7,8). However, systemic corticosteroids are used only in the rapidly progressive form of the disease nowadays (9). In the recent decades, scientists have focused on non - medical treatment options as a first - line or an adjuvant therapy . For instance, ultraviolet (uv) radiation in the form of uvb and uva as a first - line therapy in generalized vitiligo or uva as an adjuvant therapy to systemic psoralen that is believed to be increases the number of residual melanocytes (10 - 12). The latest non - medical option in treatment of vitiligo and management of melanocyte distribution is surgical intervention that has been described first by authors like behl and falabella and colleagues (13 - 15). Hair follicular transplant is one of these various surgical modalities that are followed to re - pigment the vitiligo patches . This procedure is based on the concept of existence of undifferentiated stem cells in the hair follicle, which forms a good source of melanocytes for re - pigmentation . After few weeks of grafting, the melanocytes spread to surrounding depigmented epiderm and the skin appears re - pigmented . When compared to other modalities, except the color match and tiny scars, the appearance of re - pigmentation area was much more acceptable in this method . This method is effective in focal vitiligo, vitiligo in hairy and non - glabrous areas and in those patches with leukotrichia . In this study, the hair follicular transplantation effect on re - pigmentation of affected areas in segmental vitiligo was evaluated . Study population: this registered clinical trial recruited 10 patients with documented diagnosis of segmental vitiligo who suffered from persistent form of segmental vitiligo for more than 3 years . The patients were not in the progressing phase of their disease at the time of enrollment . It is noted that a written informed consent was obtained from all of the patients and the ethics committee of our university of medical sciences approved the research project . Hair follicle autograft transplantation: after cutting hair of occipital area just by scissors and sterilization, local anesthesia was performed . 3 to 5 punch biopsies with the diameter of 5 mm were harvested from the scalp and the donor site were sutured using nylon 0.3 . Grafts were irrigated with normal saline, and separated into follicular units, which then reimplanted into the recipient sites created by 19- and 20-scalpel or nokor needles . Then the recipient sites were dressed . Patients were followed - up every two weeks for a month, then every month for 6 months evaluated for presence of re - pigmentation around the follicles . Photographs of all patients were taken before and after procedure . Statistical analysis: data presented as frequency and percentage . We analyzed data for eight male (80%) and two female (20%) patients within an age range of 21 to 43, who were enrolled into the study . Depigmented skin areas were located in the face of 4 patients (40%), extremities of 4 patients (40%) and in the trunk of the body of 2 patients (20%). Re - pigmentation was detectable in 6 cases (60%) following 2 weeks procedure . Re - pigmentation was appeared in all of the cases after 4 weeks, which continued to improve during the follow up period . Afterward, all the patients had detectable re - pigmented area of at least 2 mm and maximum of 9 mm during 6 month (fig.1,2). Follow - up results are reported in table 1 . A) skin area before treatment (b) skin area 6 month after hair follicle transplantation re - pigmented areas are presented as millimeter . Surgical interventions remain a therapeutic option for the treatment of the patients with localized form of vitiligo that have failed medical therapy . Clinically stabled segmental vitiligo with leukotrichia is one of the indications of surgical intervention . Until now, various kinds of surgical procedure have been used in treating stable vitiligo macules and patches, such as punch graft, thiersch s graft, blister - graft, full - thickness skin graft and autologous melanocyte transplants (16,17). Hair follicle transplantation was first introduced to initiate re - pigment vitiligo lesions in 1998 (18). This procedure is based on the concept of existence of undifferentiated stem cells in the hair follicle, which forms an excellent reservoir of melanocytes for re - pigmentation . Staricco (19) demonstrated that there were two types of pigment cells in the hair follicle, inactive and active melanocytes and the inactive melanocytes could migrate along with regenerated epidermis and would mature gradually . Ortonne et al (20) postulated the existence of a melanocyte reservoir, specifically located in the lower portion of human hair follicles and they proposed that re - pigmentation of vitiligo was derived from the melanocyte reservoir in the hair follicles . Cui and colleagues (21)demonstrated that during the re - pigmentation of vitiligo the number of inactive melanocytes in the outer sheath of hair follicles significantly increased and some active melanocytes appeared in the outer root sheaths, hair follicle orifices and around the perifollicular epidermis . The hypothesis of stimulation of melanocytes migration from the hair follicle reservoir by phototherapy is now a well - established fact . Melanocytes spread centrifugally from the infundibulum to the basal layer and recolonize the epidermis with active and functional melanocytes (22). Regardless of the mode of treatment, re - pigmentation in vitiligo usually begins in the perifollicular area . Transplant of hair follicle in order to stimulate re - pigmentation in vitiligo - affected areas has been reported earlier by some authors (18,23,24). Pigmentation starts appearing at 4to 5 week and continues up to 6 months or even longer(25). In this method, although the appearance of pigmentation was delayed when compared to other modalities, the color match was much more acceptable than others (table . 2) (18,23,25). Hair follicle transplantationis also more effective than the other treatment options, as transformation of depigmented hairs into the pigmented ones become evident and grafted hairs could retain the pigmentation even in cases of unresponsive or treatment - resistant (3). Our experience showed initiation and progression of re - pigmentation in all patients that was comparable and even more effective than previously reported ones . Except the undeniable limitation of the present study regarding small number of patients, it showed excellent result with high patients' satisfaction . In this method, we used small punched biopsy in order to harvest follicles from the scalp to decrease risk of scar formation and kobner effect . In addition, this method could perform in a one session and has a low cost that is much more acceptable for the patients . Given the result of the present study, autologous hair follicle harvesting through punch biopsy direct transplanting into the hairy and non - glabrous areas could effectively initiate re - pigmentation of depigmented areas in segmental vitiligo . As it may form tiny scars, it is better to apply in the hairy areas . Since segmental vitiligo is a rare form of the disease, further multi central studies or studies with and an appropriate sample size
Methods for ether synthesis are overwhelmingly based upon the attack of nucleophilic oxygen on electrophilic carbon . Our lab has been interested in expanding the scope of an umpoled strategy based upon reaction of carbanions with electrophilic oxygen . The reaction of dialkyl peroxides with carbanions, while long known, has largely remained a curiosity applied mainly to transfer of methoxyl and other unhindered alkoxides . Even a slight increase in steric bulk results in reduced yields, and reactions of di - t - butyl peroxide furnish multiple products . We became interested in a general method for selective transfer of 1, 2, or 3 alkoxides from a mixed peroxide to a carbanion . In approaching this problem, we were encouraged by evidence for the strong influence of substituent groups on peroxide reactivity . Bistrimethylsilyl peroxide efficiently transfers osime3 to a variety of organometallic reagents; curiously, tbuoosime3 transfers neither osime3 nor tbuo . Oxenoid in character, transfer lio to a variety of carbanions, and similar reactivity patterns have been observed with other metalloperoxides . The strain within bicyclic peroxides has been harnessed to facilitate reaction of more hindered peroxides; unfortunately, the presence of the scaffold limits the range of potential targets . Electronic activation in the form of peresters has been employed to achieve transfer of tertiary alkoxides to grignard reagents, but broad application is limited by the propensity of peresters to undergo c - to - o rearrangement or fragmentation to carbonyls . We were particularly intrigued by reports describing enhanced reactivity of acyclic peroxyacetals and peroxyaminals with grignard reagents . However, the reactions of peroxyaminals displayed variable regioselectivity, while reactions of a mixed ethoxy / peroxy acetal required a significant excess of the grignard reagent . In preliminary work, we discovered that tetrahydropyranyl (thp) monoperoxyacetals selectively transfer the attached or to lithiated 1,3-dithianes . We now describe investigations of reactions between carbanions and several classes of organic peroxides, with an emphasis on monoperoxyacetals . Base - promoted alkylation of hydroperoxides with primary and secondary triflates provided good yields of t - butyl / alkyl peroxides (8a11a), monoperoxyacetals (8c,8b10b, 12b, 13b(24)), and a 2-methoxyethyl peroxide (9e). Tetrahydropyranyl monoperoxyacetals (14b, 15b) were also prepared through acetalization of hydroperoxides with dihydropyran . Thermal analysis was conducted on one of the dialkyl peroxides (10a) and one of the monoperoxyacetals (10b) to provide some baseline stability data for each class . The results, detailed in supporting information, revealed that 10a and 10b are relatively stable, undergoing exothermic decomposition only once heated past 170 and 120 c, respectively . Table 2 illustrates the reactivity of t - butyl / alkyl and trialkylsilyl / alkyl peroxides toward sp organometallics . T - butyl peroxide 8a reacted with an excess of rli over a period of several hours to furnish ethers derived from selective attack on the less hindered oxygen . The corresponding trialkylsilyl peroxide (8d) reacted slowly with n - buli to furnish a large number of products (tlc). From allylsnbu3 and n - buli . Reactions with rli proceeded slowly at 78 c but were complete within minutes at 0 c . The monoperoxyacetals failed to react with grignard reagents at 78 c but were consumed rapidly at 0 c, providing ethers in yields equal or superior to those obtained with rli . Transfer of 1, 2, or 3 alkoxides occurred with similar yields; in each case; we observed only the products of regioselective displacement of othp . (a) rli (1.1 equiv), 78 c; (b) hexylmgbr (1.1 equiv), 0 c; (c) me3sich2mgcl (1.3 equiv), 0 c . Monoperoxyacetal 8b reacted readily with phli (78 c) and phmgbr (0 c) to furnish good yields of the phenyl ether (18a, scheme 2). Reaction with an alkenyl magnesium bromide or vinyl lithium, the latter generated in situ, furnished the corresponding enol ethers; the moderate yields may in part reflect decomposition during purification . Reaction of 2-thienyl lithium with primary monoperoxyacetals furnished a moderate yield of 2-alkoxythiophenes 18d and 18e, representatives of a class of molecules previously explored only to a limited extent . Thf, 78 to rt (rli) or 0 c to rt (rmgx). Alkynyllithium or alkynylmagnesium reagents failed to react with a dialkyl peroxide (8a) or a monoperoxyacetal (8b); starting materials could be recovered even after prolonged reaction periods (eq 1).1 neither monoperoxyacetals nor a dialkyl peroxide underwent intermolecular c o bond formation with enolates (eq 2) or azaenolates (eq 3); only slow decomposition was observed . The results were not entirely unexpected given our observations during tandem reactions of enolates with iodoperoxides . In a control experiment, reaction of a thf solution of 8b with stoichiometric potassium t - butoxide (eq 4) resulted in slow formation of byproducts indicative of e1cb fragmentation . The corresponding t - butyl peroxide 8a (not shown) decomposed more slowly to furnish citronellal.2 lithiated 1,3-dithianes have been widely applied as acyl anion equivalents for reaction with carbon electrophiles . In the only report of their reactivity toward peroxides, reaction with bistrimethylsilyl peroxide results in preferential transfer of a silyl group . We recently demonstrated successful reaction of thp monoperoxyacetals with lithiated dithianes to furnish s, s, o - orthoesters and derived difluoroalkyl ethers . Being curious about the influence of the peroxide electrophile on the reactivity toward inductively stabilized anions, we investigated reactions of a monoperoxyacetal, a silyl / alkyl peroxide, and a dialkyl peroxide toward a common lithiated dithiane (scheme 3). The monoperoxyacetal 10b and alkyl / silyl peroxide 10d gave similar yields of difluoroether 19, isolated following fluorodesulfurization of the initial orthoester products . In contrast, the mixed alkyl / t - butyl peroxide 10a was much less reactive . Reactions were conducted exclusively in thf; previous investigations of reactions of lithiated trithianes observed no benefit from the presence of additives (licl, mgbr2) or polar cosolvents (hmpa). Trifluoromethyl ethers are of interest as degradation - resistant structural components in agrochemicals and drug molecules . We recently reported an indirect approach to introduction of ocf3 groups via reaction of a monoperoxyacetal with a lithiated trithiane, followed by fluorodesulfurization of the resulting s, s, o - orthocarbonate, and we became interested in a more direct approach based upon reaction of a peroxide with a trifluoromethyl anion . This approach requires the targeted c o bond formation to occur more rapidly than -elimination of the nucleophile . The trifluoromethyl ether was not observed despite application of different activators, solvents (dmf, thf), or reaction temperatures (scheme 4). We also failed to detect ether formation for base - promoted reaction with trifluoroacetaldehyde hydrate, or with kcf3 generated via low temperature deprotonation of fluoroform . Formation of aldehydes derived from kornblum fragmentation of the peroxide (see eq 4) was observed in some of these reactions . Interested in whether the displacements might be more successful in the presence of a lithium cation, we investigated reactions with lithiated trihalomethanes but saw no reaction at temperatures (110 c for liccl3, 50 c for licbr3) where the reagents were stable . Lithiated ethers (-alkoxylithiums) have been employed as synthons for acyl anions and hydroxymethyl anions . As illustrated in table 3, the alkoxylithium reagent generated from a tributylstannyl mom ether (20) reacted with primary alkyl / t - butyl peroxides or a monoperoxyacetal to give moderate yields of the mixed o, o - acetals . The same reaction, when repeated with methoxypropyl peroxyacetal 8c, did not proceed to completion . The mixed acetal (21d) was isolated in low yield and accompanied by the tertiary alcohol (22) derived from trapping of acetone generated from the cleaved acetal (eq 5).5 we were interested in application of the reaction of monoperoxyacetals and organometallics as an approach to cyclopropyl ethers, a relatively challenging class of targets for which multiple approaches have been reported . In approaching this class of reactions, we were encouraged by the reported reaction of t - butyl perbenzoates with cyclopropyl magnesium bromide . We found that the reaction of monoperoxyacetals with cyclopropyl magnesium bromide furnishes an efficient approach to primary and secondary cyclopropyl alkyl ethers (table 4). In an effort to gain information about the possible role of single electron transfer in the c o bond - forming step, we investigated reactions of phenylmethylpropyl peroxides; the derived alkoxy radical undergoes cleavage to acetone and benzyl radical at 10/s (scheme 5). Silyl peroxide 24d was prepared through co(ii)-mediated peroxidation of methallyl benzene with oxygen and triethylsilane . Thp acetal 24b was prepared by deprotection of the silyl peroxide and acetalization of the intermediate hydroperoxide . Monoperoxyacetal 24b underwent rapid reaction with n - buli to furnish mainly the butyl ether (25a), accompanied by smaller amounts of the tertiary alcohol 26 and recovered starting material (table 5). The corresponding reaction with excess hexylmgbr furnished only the hexyl ether (25b) resulting from displacement of othp . In contrast, reaction of di - t - alkyl peroxide 24a with n - buli generated multiple products and only trace amounts of the ether . The reaction of silyl peroxide 24d with n - buli provided the most convincing evidence for the intermediacy of radicals in the form of a significant amount of pentylbenzene product of radical radical coupling . Conditions: n - buli (1 equiv), thf, 78 c, allow to warm to rt; hexylmgbr (2 equiv), 0 c, allow to warm to rt . A competition between a dialkyl peroxide and a monoperoxyacetal for capture of a limiting quantity of n - buli generated only the ether (27) derived from the monoperoxyacetal (eq 6).6 a competition between n - buli and phli for a limiting quantity of a monoperoxyacetal revealed a strong preference for reaction with the sp reagent (eq 7).7 in an effort to probe the basis for the enhanced reactivity of the monoperoxyacetals, we investigated c o bond formation with 2-methoxyethyl peroxide 9e . This substrate, which possesses the potential for metal chelation but lacks a peroxyacetal, provided the corresponding either in lower yields than had been observed even with simple t - butyl peroxides (eq 8).8 in an effort to gain better understanding of the reactions described above and the influence of reaction conditions on product formation, we conducted b3lyp/6 - 31+g(d, p) calculations for the simple model reaction of the transfer of methoxide from a methyl peroxide or peroxyacetal to a methyl anion (scheme 6). Our calculations focused on the impact of changing the metal from li to na and of changing the leaving group from ot - bu to othp . B3lyp/6 - 31+g(d, p), 0 k; units in kcal / mol . Thf at 298 k with the smd continuum solvent model; units in kcal / mol and enthalpies in parentheses . All four sets of conditions appear to favor the same mechanism: first, formation of a metal - peroxy complex (ii), second, passage through a four - centered transition state (ts iii), third, formation of a complex of the incipient ether with the metal alkoxide (iv), and, finally, cleavage to final products (v). (early calculations found the same mechanism for the addition of alkyl lithium reagents to carbon the calculations predict that the lithiated system is significantly more reactive than the sodiated system . This suggests that the metal ion s interaction with the peroxy moiety is primarily electrostatic; the greater charge density on the smaller li leads to greater stabilization . The presence of the thp group is found to stabilize the initial organometallic - peroxide complex (ii) and to dramatically lower the energy for insertion into the peroxide bond . An approximate treatment of thf solvation using the smd continuum model predicts the relative enthalpies of most intermediates and transition states to be several kcal / mol higher in energy than the gas - phase calculations . However, the solvation calculations predict the same trends in reactivity: the li ion and the thp substituent both significantly stabilize both the reactive complex and the transition state for insertion . Whereas reactions of dialkyl peroxides are largely limited to transfer of methoxide or primary alkoxide, monoperoxyacetals have significantly enhanced reactivity toward unstabilized carbanions, transferring primary, secondary, or tertiary alkoxides through highly regioselective attack on the nonacetal oxygen of the peroxide . In contrast to peresters, monoperoxyacetals appear to promote alkoxide transfer without significantly destabilizing the peroxide . Reaction is observed between monoperoxyacetals and sp and sp rli and rmgx reagents at 78 c (organolithium reagents) or 0 c (grignard reagents). Both acyclic (2-methoxyprop-2-yl) and cyclic (thp) peroxyacetals offer good reactivity; however, the acyclic peoxyacetals sometime generate side products derived from liberation of a reactive carbonyl group in the presence of the organometallic . The monoperoxyacetals do not undergo intermolecular reactions with enolates or similar stabilized carbanions, paralleling earlier observations from our lab . The monoperoxyacetals also fail to react with metalated alkynes, an interesting outcome given the reported reactivity of lithiated hydroperoxides toward carbanions, as well as our observation of successful reaction of peroxyacetals with inductively stabilized anions such as lithiated dithianes and lithiated ethers . However, the lack of reactivity toward sp carbanions is consistent with results observed with bistrimethylsilyl peroxide, which undergoes mainly silyl exchange with lithiated alkynes . Several mechanisms have been discussed with regard to the reaction of dialkyl peroxides with unstabilized organometallics . While early results with unhindered peroxides were consistent with sn2-type attack on the o o bond, reactions of di - t - alkyl peroxides have been reported to proceed via intermediate alkoxy radicals . Our work found no evidence for radical intermediates in attack of organolithium reagents on thp peroxyacetals . If the reactions involve neither simple displacement nor cleavage to alkoxy radicals, then what? Lithiated peroxides are often thought to react with nucleophiles through oxenoid - type insertion into the c li bond; attack on alkenyl lithium and cyclopropyl lithium appears to occur without loss of stereochemistry . However, whereas the transfer of lio from lithiated hydroperoxides to organolithium reagents has been calculated to involve in - line arrangement of the carbanion, the lithiated oxygen, and the departing oxygen, our calculations suggest side - on insertion of the rli into the lewis acid activated o o bond . This theoretical result is supported by the similar yields obtained for transfer of primary, secondary, and tertiary alkoxides, a result which would seem to rule out an sn2-type attack on the backside of the breaking o o bond . Calculations predict, and experiments confirm, that the presence of the acetal group significantly lowers the energy for insertion of unstabilized organometallics and directs the reaction toward exclusive transfer of the nonanomeric oxygen . The bidentate coordination available to the organometallic / lewis acid is not sufficient to explain the results, as evidenced by the poor reactivity of the methoxyethyl peroxide . However, the interaction of lewis acids with peroxyacetals and ozonides (1,2,4-trioxolanes) has been previously demonstrated to activate both c o and o o bonds . In conclusion, we have shown that monoperoxyacetals, readily prepared and easily handled derivatives of hydroperoxides, enable highly efficient intermolecular transfer of alkoxide to unstabilized organolithium and organomagnesium reagents along with some inductively stabilized organolithium species . The peroxyacetal activating group promotes highly selective transfer of the nonanomeric or, regardless of steric bulk . The bond - forming process, which appears to involve metal - promoted insertion into the o o linkage, appears to be mechanistically distinct from similar reactions of alkyl or alkyl silyl peroxides . The new methodology offers a new tool with which to approach ethers, including some poorly accessible through existing methods . Reagents and solvents were used as supplied commercially, except for dcm and thf, which were distilled from cah2 and na / ph2co, respectively . Thin - layer chromatography (tlc) was performed on 0.25 mm hard - layer silica g plates visualized with a uv lamp or by staining: 1% ceric sulfate and 10% ammonium molybdate in 10% h2so4 (general stain, after heating); 3% vanillin in 3% h2so4 in etoh (general stain, after heating); or a 1% n, n-dimethyl - p - phenylenediamine in 1:20:100 acetic acid / water / methanol (specific for peroxides). Unless otherwise noted, nmr spectra were acquired in cdcl3; h spectra are reported as chemical shift (multiplicity, j couplings in hz, number of protons). Ir spectra were recorded as neat films on a zrse crystal; selected absorbances are reported in cm . Abbreviations: hexane = hex; ea = ethyl acetate; thf = tetrahydrofuran; tbdms (or tbs) = tert - butyldimethylsilyl; thp = tetrahydropyranyl . Optimized geometries, harmonic vibrational frequencies, and electronic energies of all structures in scheme 6 were obtained with the b3lyp level of density functional theory and the 6 - 31+g(d, p) basis set . All minima reported (structures i, ii, iv, and v) contain all real frequencies, and all transition states (structures ts iii) contain one imaginary frequency . Intrinsic reaction coordinate calculations confirmed that each transition state connected a metal - peroxy complex ii and the corresponding metal alkoxide - ether complex iv . The relative electronic energies were corrected by the differences in zero - point vibrational energy scaled by 0.9806 . Solution - phase enthalpies were determined as follows: the thermal corrections to the electronic energies were calculated based on the b3lyp/6 - 31+g(d, p) geometries and harmonic frequencies . Solvation corrections were based on single - point energy calculations using the continuum smd model of truhlar and co - workers . [75 - 91 - 2], 5.5 m in decane, was used as purchased . [4676 - 84 - 0] was synthesized using a modified version of a known procedure . To a 0 c solution of 50 wt% aq . H2so4 (0.1 ml) the mixture was stirred for 10 min, after which 3,4-dihydro-2h - pyran (4.94 g, 58.8 mmol) was added over a period of 3 min . The reaction was stirred for 1 h at 0 c and then diluted with sat . The solution was extracted with ether (150 ml), and the organic layer was washed with aq . Sat . The residue obtained was purified by silica gel flash chromatography using a gradient of 425% ea / hex to afford the hydroperoxide as a thick and colorless oil (4.60 g, 66%). [10027 - 74 - 4] was prepared as a colorless oil (82%, 878 mg) from 2,3-dimethyl-2-butene (10.0 mmol, 841 mg) using a known procedure.rf = 0.3 (15% ea: hex). [123369 - 58 - 4] was prepared by alkylation of a 1,1-dihydroperoxycyclodecane, followed by hydrolysis of the resulting bisperoxyacetal . A solution of 3,7-dimethyloct-6-en-1-ol (0.936 g, 6 mmol) in dry dcm (18 ml) was cooled to 0 c, and triflic anhydride (1.4 equiv) and 2,6-lutidine (1.5 equiv) were sequentially added via syringe . The reaction was stirred for 15 min at 0 c and then diluted with ice - cold hexane (40 ml). Khso4 (40 ml), and the separated aqueous layer was extracted with another portion of cold hex (25 ml). The combined organic layers were dried over na2so4 and concentrated on a rotary evaporator (bath temperature 10 c). The residue was subjected to 0.5 mmhg for approximately 5 min to generate nearly pure 3,7-dimethyloct-6-en-1-yl trifluoromethanesulfonate (1.693 g, 98%) as a light brown oil, which was placed in a 20 c freezer and used within an hour: rf = 0.59 (10% ea / hex); h nmr: 5.115.07 (m, 1h), 4.614.57 (m, 2h), 2.061.96 (m, 2h), 1.931.85 (m, 1h), 1.721.68 (m, 1h), 1.70 (s, 3h), 1.661.64 (m, 1h), 1.62 (s, 3h), 1.421.32 (m, 1h), 1.291.20 (m, 1h), 0.96 (d, j = 6.3 hz, 3h); c nmr: 131.8, 123.9, 118.5 (q, j = 323 hz), 76.2, 36.6, 36.0, 28.6, 25.6, 25.1, 19.0, 17.6 . To a 0 c solution of cyclododecanone-1,1-dihydroperoxide (2.196 g, 9.469 mmol, synthesized as previously described) in dry thf (50 ml) was added kotbu (2 equiv), followed by the triflate described above (6.0 g, 20.8 mmol). The reaction mixture was stirred until the starting material was no longer visible (tlc, 20 min). The reaction mixture was then quenched with water (25 ml) and extracted with ea (50 ml 2). The residue was purified by silica chromatography using 1% ea / hex to furnish 1,1-bis-(3,7 dimethyl-6-octenylperoxy) cyclododecane (2.84 g, 59%) as a colorless oil: rf = 0.75 (10% ea: hex); h nmr: 5.10 (m, 2h), 4.173.99 (m, 4h), 2.071.91 (m, 4h), 1.721.64 (m, 9h), 1.69 (s, 3h), 1.61 (s, 3h), 1.581.53 (m, 7h), 1.481.28 (m, 20h), 1.241.15 (m, 2h), 0.92 (d, j = 6.5, 6h); c nmr: 131.2, 124.6, 113.1, 73.4, 37.1, 34.6, 29.7, 27.0, 26.1, 25.7, 25.4, 22.3, 21.9, 19.6, 19.4, 17.6; hrms (tofms - es): calcd for c32h60o4na (m + na) 531.4389 found: 531.4373; ir: 2926, 2850, 1445, 1052 . To a room temperature solution of the bisperoxyacetal (1.01 g, 2 mmol) in thf (20 ml) was added a solution of 50% aq . H2so4 (1.8 mmol, 6 equiv), and the reaction mixture was stirred at 55 c until starting material was nearly consumed (tlc, 13 h). Na2co3 (25 ml), and the resulting mixture was extracted with ea (30 ml 2). The combined organic layers were dried over na2so4 and concentrated on a rotary evaporator . The crude product was purified by silica chromatography using 13% ea / hex to furnish hydroperoxide 4 as a colorless oil (0.364 mg, 53%). The molecule has previously been reported without characterization: rf = 0.4 (10% ea / hex); h nmr: 8.53 (s, 1h), 5.09 (m, 1h), 4.104.00 (m, 2h), 2.061.93 (m, 2h), 1.731.63 (m, 1h), 1.68 (s, 3h), 1.60 (s, 3h), 1.541.35 (m, 2h), 1.331.14 (m, 2h), 0.91 (d, j = 6.5, 3h); c nmr: 131.3, 124.6, 75.5, 37.1, 34.3, 29.5, 25.7, 25.4, 19.5, 17.6 . Ir: 36173101 (broad peak), 2915, 1455, 1377, 1053 (mass spec not attempted due to volatility). [4952 - 03 - 8] was prepared through a two - step procedure . (cas 634600 - 89 - 8) was prepared through a modification of a reported procedure . 1-methyl cyclohexene (0.96 g, 10 mmol) and et3sih (2.32 g., 20 mmol) were sequentially added to a solution of co(acac)2 (1 mmol) in ethanol (35 ml). The reaction mixture was placed under an atmosphere of o2 (balloon) until no starting material was observed (tlc, 14 h). The residue obtained upon concentration in vacuo was purified by silica gel chromatography using 18% ea: hex to furnish the 1-methyl-1-cyclohexyl triethylsilyl peroxide as a as colorless oil (0.844 g, 34%): rf = 0.7 (10% ea / hex). To a solution of the triethylsilylperoxide (0.78 g, 3.19 mmol) in thf (10 ml) was added tetra n - butyl ammonium fluoride (1 m solution in thf, 1.2 equiv). The reaction was stirred at room temperature until no starting material could be observed (tlc, 5 min). The residue obtained upon concentration in vacuo was diluted with hexane (30 ml) and washed with water (10 ml). The residue was purified by short - column (3) flash chromatography with 5% ea / hex (two iterations were required to remove silanol / siloxane byproducts) to furnish the hydroperoxide 5 as a colorless oil (0.266 g, 64%). Although the hydroperoxide is commercially available, nmr data are difficult to access and are provided here for convenience: rf = 0.4 (10% ea: hex); h nmr: 7.34 (s, 1h), 1.801.74 (m, 2h), 1.631.53 (m, 2h), 1.511.38 (m, 5h), 1.341.29 (m, 1h), 1.25 (s, 3h); c nmr: 81.7, 34.4, 25.6, 23.8, 22.2, 6.55, 5.76 . [60956 - 33 - 4] was prepared using a route analogous to that employed for hydroperoxide 4 . 3-phenylpropyl trifluoromethanesulfonate [66950 - 73 - 0] was prepared (1.34 g, quant .) As a light brown oil from 3-phenylpropanol (0.681 g, 5 mmol) using the procedure described above: rf = 0.5 (10% ea / hex). Other spectral data matched those in a previous report . Using a similar procedure as described earlier, cyclododecanone 1,1-dihydroperoxide (1.05 g, 4.545 mmol) was reacted with the 3-phenylpropyl triflate (2.6 g, 10 mmol) to furnish the bisperoxyacetal (1.4381 g, 67%) as a colorless oil: rf = 0.6 (15% ea / hex); h nmr: 7.327.29 (m, 4h), 7.237.21 (m, 6h), 4.11 (t, j = 6.4, 4h), 2.73 (t, j = 7.5, 4h), 2.021.95 (m, 4h), 1.741.70 (m, 4h), 1.561.46 (m, 4h), 1.451.31 (m, 14h); c nmr: 141.7, 128.4, 128.3, 125.8, 113.3, 74.2, 32.3, 29.5, 27.1, 26.1, 22.3, 22.0, 19.4; ir: 2914, 2845, 1462, 1053 hydrolysis of the bisperoxyacetal (1.35 g, 2.88 mmol) by a similar procedure as described above furnished hydroperoxide 6 as a colorless oil (0.725 g, 82%): rf = 0.57 (20% ea / hex). The title compound was prepared by an analogous strategy as described for hydroperoxides 4 and 6 . 10-phenyldecyl trifluoromethanesulfonate (1.098 g, quant .) Was prepared from 10-phenyldecanol (0.70 g, 3 mmol) as a light brown oil: rf = 0.65 (10% ea / hex); h nmr: 7.337.28 (m, 2h), 7.227.18 (m, 3h), 4.56 (t, j = 6.5 hz, 2h), 2.63 (t, j = 7.7 hz, 2h), 1.85 (m, 2h), 1.691.59 (m, 2h), 1.461.33 (m, 12h); c nmr: 142, 128.4, 128.2, 125.5, 118.6 (q, j = 320 hz), 77.7, 35.9, 31.5, 29.4, 29.37, 29.30, 29.27, 29.22, 28.8, 25.0 . Hrms (esi): calcd for c17h25f3nao3 s (m + na) 389.1374, found 389.1369 . Using a similar procedure as described above, reaction of 1,1-dihydroperoxycyclododecanone (0.293 g, 1.26 mmol) and the 10-phenyl decyl triflate (1.02 g, 2.787 mmol) was used to prepare 1,1-bis-(10-phenyldecyldioxy)cyclodecanone: (0.834 g, 99%) as a colorless oil: rf = 0.74 (15% ea / hex); h nmr: 7.327.29 (m, 4h), 7.217.19 (m, 6h), 4.09 (t, j = 6.6, 4h), 2.62 (t, j = 7.7, 4h), 1.721.59 (m, 12h), 1.371.30 (m, 42h); c nmr: 142.9, 128.4, 128.2, 125.5, 113.1, 75.0, 36.0, 31.5, 29.57, 29.53, 29.48, 29.36, 27.8, 27.0, 26.2, 26.1, 22.3, 21.9, 19.4 . Hrms (tof - ms - es+): calcd for c44h72o4na (m + na) 687.5328 found 687.5300; ir: 2914, 2845, 1462, 1053 . Using a procedure similar to that described above, hydrolysis of the bis-(10-phenyldecyl)peroxyacetal (0.755 g, 1.14 mmol) furnished 10-phenyldecyl hydroperoxide as a colorless oil (0.243 g, 64%): rf = 0.55 (15% ea / hex); h nmr: 8.04 (t, j = 6.5, 1h), 7.337.28 (m, 2h), 7.227.20 (m, 3h), 4.04 (t, j = 6.6, 2h), 2.63 (t, j = 7.7, 2h), 1.681.61 (m, 4h), 1.32 (m, 12h); c nmr: 142.9, 128.4, 128.2, 125.5, 76.6, 36.0, 31.5, 29.5, 29.4, 29.3, 27.5, 25.9; hrms (tof - ms - ei+): calcd for c16h25o (m oh) 233.1905; found: 233.1920; ir: 3390, 2922, 2852, 1453, 696 the hydroperoxide (10 mmol in a minimum amount of thf) was added under nitrogen to a 0 c solution of kotbu (10 mmol) in dry thf (50 ml). The reaction mixture was stirred for 3 min, after which was added preformed alkyl triflate (10 mmol, neat from syringe, remaining triflate rinsed into the reaction using 1 ml of thf). The reaction was stirred for 15 min at the same temperature and then quenched with water (20 ml). The mixture was extracted with 10% ea / hex (50 ml 2), and the organic layer was dried over na2so4 . The residue obtained upon concentration was purified by flash silica chromatography using 1% ea / hex . Acetalization of alkyl hydroperoxides to form tetrahydropyranyl (thp) monoperoxyacetals employed a modification of a reported procedure . A mixture of alkyl hydroperoxide (1.0 mmol) and 2,3-dihydropyran (1.0 mmol) was cooled to 0 c, after which was added 0.05 ml of a solution of 10% h2so4 in thf . The stirred reaction mixture was allowed to warm to rt over a period of 45 min . The reaction was diluted with 10% ether / hex (15 ml), and the resulting solution was washed with water (2 ml). The separated organic layer was dried over na2so4 and concentrated under reduced pressure . Dialkyl peroxide synthesis using silver oxide was based upon a modification of reported procedures . The alkyl hydroperoxide (1.0 mmol) was added to a suspension (ea, 10 ml) of freshly prepared silver oxide (1.2 mmol). Alkyl halide (1.0 mmol) was added, and the reaction was stirred at rt until no starting material could be observed (tlc, approximately 410 h). The reaction mixture was filtered through a small pad of celite, and the residue was washed with ea (1020 ml). The combined filtrates were concentrated, and the residue was purified by silica gel chromatography using 13% ea / hex . Trialkylsilylation was performed based upon a reported procedure, except for a different order of addition and the use of water rather than aqueous acid for the wash . To a room temperature solution of alkyl hydroperoxide (1.0 mmol) in dmf (2 ml) was added tert - butyl dimethylsilyl chloride (1.2 mmol), followed by imidazole (1.4 mmol). The reaction was stirred for 1 h and then diluted with water (10 ml). The hexane extracts (1 50, 1 10 ml) were dried over na2so4 and concentrated under reduced pressure . [1592933 - 34 - 0]: using method a (above), t - butyl hydroperoxide (5.5 m in decane, 0.588 ml, 3.24 mmol) was reacted with 3,7-dimethyl-6-octenyl triflate (described above in relation to preparation of peroxide 4, 1.025 g, 3.559 mmol) to furnish 8a as a colorless oil (0.511 g, 69%); rf = 0.6 (10% ea / hex). Spectral data matched those in literature reports . Using method a, thp hydroperoxide 1 (0.63 g, 5.34 mmol) was reacted with 3,7-dimethyl-6-octenyl triflate (1.69 g, 5.87 mmol) to furnish peroxide 9a as a colorless oil (0.854 g, yield: 62%) rf = 0.42 (10% ea / hex); h nmr: 5.15 (t, j = 3.5 hz, 1h), 5.10 (m, 1h), 4.194.00 (m, 2h), 4.02 (m, 1h), 3.663.61 (m, 1h), 1.98 (m, 2h), 1.781.71 (m, 2h), 1.701.65 (m, 1h), 1.68 (s, 3h), 1.641.53 (m, 5h), 1.60 (s, 3h), 1.491.42 (m, 1h), 1.411.32 (m, 1h), 1.231.1 (m, 1h), 0.92 (d, j = 6.6 hz, 3h); c nmr:: 131.2, 124.7, 100.8, 73.7, 62.5, 37.1, 34.6, 29.6, 27.96, 25.7, 25.4, 25.1, 19.7, 19.5, 17.6 . Hrms (esi tof) calcd for c15h28nao3 (m + na): 279.1931; found: 279.1934; ir: 2923, 2861, 1440, 1040 . Using method a, hydroperoxide 3 (0.674 g, 6.35 mmol, 1.5 equiv) was reacted with 3,7-dimethyl-6-octenyl triflate (1.21 g, 4.2 mmol) to furnish 8c as a colorless oil (0.627 g, yield: 61%); rf = 0.4 (10% ea / hex); h nmr: 5.1 (m, 1h), 4.114.01 (m, 2h), 3.33 (s, 3h), 2.061.91 (m, 2h), 1.711.64 (m, 1h), 1.69 (s, 3h), 1.61 (s, 3h), 1.621.54 (m, 1h), 1.471.31 (m, 2h), 1.40 (s, 6h), 1.241.10 (m, 1h), 0.91 (d, j = 6.5 hz, 3h); c nmr131.2, 124.7, 104.5, 73.4, 49.2, 37.1, 34.6, 29.6, 25.7, 25.4, 22.76, 22.74, 19.5, 17.6; hrms (tof - ms - es+) calcd for c14h28nao3 (m + na): 267.1931; found: 267.1926; ir: 2954, 2874, 1457, 836 . Using method d, alkyl hydroperoxide 4 (0.22 g, 1.279 mmol) was silylated with t - butyldimethylsilyl chloride (tbs - cl) to furnish 8d as a colorless oil (0.212 g, 58%): rf = 0.75 (10% ea / hex); h nmr: 5.12 (m, 1h), 4.063.97 (2h), 2.061.91 (m, 2h), 1.69 (s, 3h), 1.671.59 (m, 1h), 1.61 (s, 3h), 1.591.51 (m, 1h), 1.461.30 (m, 2h), 1.221.15 (m, 1h), 0.95 (s, 9h), 0.91 (d, j = 6.6), 0.17 (s, 6h); c nmr: 131.2, 124.6, 75.3, 37.1, 34.5, 29.6, 26.1, 25.7, 25.4, 19.6, 18.1, 17.6, 5.9; hrms (tof - ms - ei+) calcd for c16h34nao2si (m + na): 309.2220; found: 309.2218; ir: 2928, 2857, 1461, 834 . [419568 - 76 - 6]: using method a, tert - butyl hydroperoxide (1.8 ml, 5.06.0 m in decane, 10 mmol) was reacted with 3-phenyl propyl triflate (2.68 g, 10 mmol) to furnish 9a as a colorless liquid (1.5151 g. 73%). [1630792 - 49 - 2]: using method a, thp hydroperoxide 1 (1.18 g, 10 mmol) was reacted with 3-phenylpropyl triflate (2.68 g, 10 mmol) to furnish peroxide 9b as a colorless oil (1.937 g, 82%); rf = 0.66 (15% ea / hex). 2-methoxyethyl trifluoromethanesulfonate [112981 - 50 - 7] was prepared (2.08 g, quant) from 2-methoxyethanol (760 mg, 10.0 mmol) using the procedure described above and used without purification . Spectral data matched those reported.rf = 0.5 (30% ea / hex). Using method a, alkyl hydroperoxide 6 (0.70 g, 4.6 mmol) was reacted with 2-methoxyethyl trifluoromethanesulfonate (0.956 g, 4.6 mmol) to furnish 9e as a colorless oil (0.723 g, 74%): rf = 0.62 (20% ea / hex); h nmr: 7.327.27 (2h), 7.227.18 (3h), 4.15 (m, 2h), 4.05 (t, j = 6.4, 2h), 3.62 (m, 2h), 3.40 (s, 3h), 2.71 (t, j = 7.8, 2h), 2.011.93 (2h); c nmr: 141.6, 128.46, 128.39, 125.9, 73.59, 73.54, 69.8, 59.1, 32.2, 29.4; hrms (tof - ms - ci+) calcd for c12h18o3na [m + na]: 233.1154; found: 233.1152; ir: 2924, 1496, 1453, 745 . Using method a, t - butyl hydroperoxide (1.41 ml, 5.06.0 m in decane, 7.76 mmol) was reacted with 10-phenyldecyl triflate (3.125 g, 8.54 mmol) to furnish 10a as a colorless oil (2.42 g, 92%). Rf = 0.73 (10% ea / hex); h nmr: 7.337.20 (m, 2h), 7.227.17 (m, 3h), 3.96 (t, j = 6.7 hz, 2h), 2.62 (t, j = 7.8 hz, 2h), 1.691.56 (m, 4h), 1.321.28 (m, 12h), 1.27 (s, 9h); c nmr:: 142.9, 128.4, 128.2, 125.5, 80.0, 75.1, 36.0, 31.5, 29.5, 29.3, 27.8, 26.3, 26.2 . Hrms (esi tof): calcd for c20h34nao2 (m + na): 329.2451; found: 329.2470; ir: 2924.8, 2853.72, 1361.5, 697 . [1630792 - 52 - 7]: using method a, thp hydroperoxide 2 (0.20 g, 1.77 mmol) was reacted with 10-phenyldecyl triflate (0.65 g, 1.775 mmol) to furnish 10b as a colorless oil (0.448 g, 75%); rf = 0.42 (10% ea / hex). Spectral properties matched those in a literature report . Using method d, alkyl hydroperoxide 7 (0.22 g, 0.89 mmol) was converted into the corresponding silyl peroxide 10d, which was obtained as a colorless oil (0.267 g, 82%): rf = 0.8 (10% ea / hex); h nmr: 7.327.28 (m, 2h), 7.227.19 (m, 3h), 3.99 (t, j = 6.6 hz, 2h), 2.63 (t, j = 7.7 hz, 2h), 1.661.56 (m, 4h), 1.32 (m, 12h), 0.97 (s, 9h), 0.19 (s, 6h); c nmr: 142.9, 128.4, 128.2, 125.5, 76.6, 36.0, 31.5, 29.5, 29.3, 27.7, 26.2, 26.1, 18.1, 5.8; hrms (esi tof) calcd for c22h40nao2si (m + na): 387.2690; found: 387.2677; ir: 2925, 2854, 1462, 1248 . [38375 - 34 - 7]: 1-octyl trifluoromethanesulfonate [71091 - 89 - 9] was prepared (3.08 g, 98%) as a light brown oil from 1-octanol (1.56 g, 12 mmol) using the general procedure described above . The h nmr spectrum matched data previously reported: rf = 0.52 (10% ea / hex); h nmr: 4.55 (t, j = 6.54, 2h), 1.84 (m, 2h), 1.461.40 (m, 2h), 1.361.29 (m, 8h), 0.90 (t, j = 6.9, 3h); c nmr: 118.2 (q, j = 320.1 hz), 76.7, 31.6, 29.2, 28.9, 28.8, 25.0, 22.5, 14.0 . T - butyl hydroperoxide (1.81 ml, 5.5 m in decane, 10 mmol) was reacted with octyl triflate (2.88 g, 11 mmol) to furnish 11a as a colorless oil (1.78 g, 88%): rf = 0.79 (10% ea / hex). The molecule has been previously reported without spectral characterization: h nmr: 3.93 (t, j = 6.7 hz, 2h), 1.631.55 (m, 2h), 1.341.26 (m, 10h), 1.25 (s, 9h), 0.88 (t, j = 6.9 hz, 3h); c nmr: 79.9, 75.1, 31.8, 29.4, 29.2, 27.8, 26.3, 26.2, 22.6, 14.0; hrms (esi - tof - ms) calcd for c12h26nao2 (m + na): 225.1825; found: 225.1840; ir: 2924, 2856, 1361, 1197 . [1630792 - 51 - 6]: decyl trifluoromethanesulfonate [53059 - 89 - 5] was prepared (3.1 g, quant = 2.9 g) as a light brown oil from 1-decanol (1.58 g, 10.0 mmol) using the procedure described above . Spectra closely matched those in previous reports: rf = 0.52 (10% ea / hex); h nmr: 4.55 (t, j = 6.5 hz, 2h), 1.84 (m, 2h), 1.43 (m, 2h), 1.361.28 (m, 12h), 0.9 (t, j = 6.8 hz, 3h); c nmr: 118.6 (q, jc f = 320 hz), 77.7, 31.8, 29.4, 29.3, 29.2, 28.8, 25.0, 22.6, 14.0 . Using method a, 2-tetrahydropyranyl hydroperoxide 2 (1.18 g, 10 mmol) was reacted with decyl triflate (2.98 g, 10 mmol) to furnish peroxide 12b as a colorless oil (2.05 g, 79%); rf = 0.56 (15% ea / hex). [1630792 - 50 - 5]: 2-octyl trifluoromethanesulfonate [58864 - 30 - 5] was prepared (3.58 g, estimated 91%) as a light brown oil from 2-octanol (2.0 g, 15 mmol) using the procedure described above . Rf = 0.3 (10% ea / hex)., hydroperoxide 2 (0.595 g, 5.01 mmol) was reacted with 2-octyl triflate (1.47 g, 5.61 mmol) to furnish 13b as a colorless oil (0.808 g, 70%) rf = 0.61 (20% ea / hex). [685877 - 38 - 7]: using method b, acetalization of 2-tetrahydropyranyl hydroperoxide 2 (0.35 g, 2.97 mmol) with dihydropyran (0.249 g, 2.97 mmol) furnished peroxide 14b as a colorless oil (0.493 g, 84%). This molecule has been previously prepared without nmr characterization: rf = 0.50 (10% ea / hex); h nmr: 5.22 (m, 2h), 4.09 (m, 1h), 4.00 (m, 1h), 3.633.60 (m, 2h), 1.761.74 (m, 4h), 1.661.53 (m, 8h); c nmr: 101.8, 100.2, 62.5, 62,1, 27.9, 27.7, 25.1, 25.0, 19.6, 19.4 . Using method b, hydroperoxide 5 (0.266 g, 2.04 mmol) was reacted with dihydropyran (0.171 g, 2.04 mmol) to furnish 15b as a colorless oil (0.326 g, 74%). Rf = 0.50 (10% ea / hex); h nmr: 5.04 (broad triplet, 1h), 4.02 (m, 1h), 3.60 (m, 1h), 1.75 (m, 5h), 1.681.53 (m, 6h), 1.471.35 (m, 6h), 1.26 (s, 3h); c nmr: 101.0, 81.5, 62.7, 35.0, 27.8, 25.7, 25.1, 24.3, 22.5, 22.3, 20.0; hrms (esi tof) calcd for c12h22nao3 (m + na): 237.1461; found: 237.1469; ir: 2931, 2852, 1443, 962 . Using a known procedure, cross - metathesis of 1-octene (6.72 g, 4.8 mmol) and allyl bromide (1.45 g, 12 mmol) in the presence of the grubbs ii catalyst furnished 1-bromo-2-undecene (1.109 g, 80%) as a colorless oil consisting of a 84:16 e / z mixture using a known procedure . The portions of the spectra corresponding to the major (e) isomer closely matched a previous report [67952 - 61 - 8]:rf = 0.8 (hexane); h nmr: (both isomers, partial integrals given): 5.825.58 (m, 2h), 4.01 (d, j = 8.3 hz, 0.32 h), 3.96 (d, j = 8.2 hz, 1.68 h), 2.14 (m, 0.32h), 2.07 (m, 1.68), 1.401.35 (m, 2h), 1.341.28 (m, 10h),5.82 0.89 (t, j = 6.8 hz, 3h); c nmr: (major isomer): 136.7, 126.2, 33,6, 32.0, 31.9, 29.4, 29.2, 29.1, 28.8, 22.7, 14.1 . Using method c, hydroperoxide 2 (0.118 mg, 1 mmol) was reacted with an e / z - mixture of 2-undecenyl bromide (0.232 g, 1.0 mmol) to furnish peroxide 16b as a colorless oil (0.197 mg, 73%) consisting of an inseparable 84:16 e / z mixture: rf = 0.42 (10% ea / hex); h nmr: 5.825.74 (m, 1h), 5.695.55 (m, 1h), 5.16 (m, 1h), 5.64 (d, j = 6.8 hz, 0.24 h), 4.51 (d, j = 6.8 hz, 1.76 h), 4.01 (m, 1h), 3.643.60 (m, 1h), 2.132.02 (m, 2h), 1.731.68 (m, 2h), 1.651.53 (m, 4h), 1.381.35 (m, 2h), 1.311.26 (m, 10h), 0.87 (t, j = 6.8 hz, 3h); c nmr: 137.8, 123.7, 100.8, 76.2, 62.4, 32.3, 31.8, 29.4, 29.2, 29.1, 28.8, 27.8, 25.1, 22.6, 19.6, 14.0 . Hrms (tof - ms - es+) calcd for c16h30nao3 (m + na): 293.2087; found: 293.2087; ir: 2923, 2851, 1202, 962 . The alkyllithium (0.55 mmol, 1.1 equiv, typically as a solution in hex) was added to the solution of peroxide (0.50 mmol) in dry thf (3 ml) at 78 c, and the reaction was stirred for 15 min . For thp monoperoxyacetals, the reaction was brought to room temperature for 15 min and then quenched with water (2 ml); for other peroxides, the reaction was allowed to stir at room temperature for the time indicated . The mixture was extracted with 20% ether in hex (1 25, 1 5 ml), and the combined organic layers were dried over na2so4 and concentrated . The residue was purified by silica gel chromatography (0.5 4 in . Note: for volatile ethers, column fractions were carefully concentrated on a rotary evaporator at 20 c bath temperature and then briefly (1 min) concentrated on high vacuum while held in an ice bath . For reaction with grignard reagents, a solution of the alkyl magnesium bromide (0.55 mmol, 1.1 equiv) in diethyl ether was added to a 0 c solution of the thp monoperoxyacetal (0.5 mmol) in dry thf (3 ml). After 15 min, the reaction was quenched with water (2 ml) and extracted with 20% ether / hex (1 25, 1 5 ml). The combined organic layers were dried over na2so4, and the concentrated residue was purified by silica gel chromatography (0.5 4 in ., column fractions were concentrated on a rotary evaporator at 20 c bath temperature and subsequently subjected to high vacuum (<1 mm) for 1 min while placed in an ice bath . [71077 - 30 - 0]: using the general procedure described above, thp monoperoxyacetal 8b (128 mg, 0.5 mmol) was reacted with n - buli (0.34 ml, 1.6 m in hex, 0.55 mmol) to furnish ether 17a as a colorless oil (86 mg, 81%). The same procedure, when applied to dialkyl peroxide 8b or 2-methoxypropyl peroxyacetal 8c furnished 17a in 70% and 87% yields, respectively . Spectral data matched those in previous reports.rf = 0.6 (5% ea / hex). Using the general procedure for grignard - promoted etherification described above, monoperoxacetal 8b (128 mg, 0.5 mmol) was reacted with n - hexyl magnesium bromide (0.27 ml, 2 m in diethyl ether, 0.55 mmol) to furnish hexyl ether 17b as a colorless oil (106 mg, 88%). The same product could be obtained from dialkyl peroxide 8a except in 42% yield: rf = 0.70 (10% ea / hex); h nmr: 5.11 (m, 1h), 3.473.38 (m, 4h), 2.081.94 (m, 2h), 1.69 (s, 3h), 1.651.53 (m, 4h), 1.61 (s, 3h), 1.451.31 (m, 8h), 1.231.11 (m, 1h), 0.910.88 (m, 6h); c nmr: 131.1, 124.8, 71.0, 69.1, 37.2, 36.7, 31.7, 29.7, 29.6, 25.9, 25.7, 25.4, 22.6, 19.6, 17.6, and 14.0 . Hrms (tof - ms - ei) calcd for c16h32o: 240.2453; found: 240.2453; ir: 2954, 2927, 2858, 1455, 1376, 1112 . [139694 - 24 - 9]: to a 0.2 m solution of peroxide 8a (229 mg, 1.0 mmol) in thf was added allyltributyl tin (0.62 ml, 2.0 mmol). The solution was cooled to 78 c, and n - buli (2.5 mmol, 1.0 ml, 2.5 m in hex) was added dropwise . After 30 min, the reaction was quenched with 10 ml of water and extracted with ether . The residue was then purified by column chromatography (2.5% ea / hex) to yield 137 mg (70%) of 17c as a colorless oil . The h nmr spectrum matched the listing in a previous report.rf = 0.25 (5% ea / hex); h nmr: 0.92 (d, 3h, j = 6.7), 1.18 (m, 1h), 1.39 (m, 1h), 1.521.68 (s at 1.62 overlapping unresolved signal, 6h), 1.70 (s, 3h), 2.00 (m, 2h), 3.48 (m, 2h), 3.98 (dt, 2h, j = 5.6, 1.4), 5.145.08 (m 1h), 5.19 (app . Dq, hcis, j = 10.6, 1.3, 1h), 5.29 (app . Dq, htrans, j = 17.2, 1.6, 1h), 5.9 (m, 1h); c nmr: 17.6, 19.6, 25.5, 25.7, 29.6, 36.7, 37.2, 68.7, 71.8, 116.7, 124.8, 131.2, 135.1 . [436141 - 44 - 5]: using the general procedure for etherification described above, peroxyacetal 8b (128 mg, 0.5 mmol) was reacted with t - buli (0.32 ml 1.7 m in pentane, 0.55 mmol) to furnish 17d as a colorless oil (55 mg, 51%). Spectral data were nearly identical to those previously described: rf = 0.6 (5% ea / hex); h nmr: 5.11 (m, 1h), 3.423.31 (m, 2h), 2.061.91 (m, 2h), 1.69 (s, 3h), 1.61 (m, 3h), 1.591.51 (m, 2h), 1.391.27 (m, 2h), 1.19 (s, 9h), 1.171.10 (m, 1h), 0.90 (d, j = 6.5 hz, 3h); c nmr: 131.0, 124.9, 72.3, 59.7, 37.7, 37.2, 29.6, 27.5, 25.7, 25.4, 19.6, 17.6 . Using the general procedure for etherification described above, peroxide 9b (118 mg, 0.50 mmol) was reacted with 2-trimethylsilyl methyl magnesium chloride (0.65 ml, 1 m in ether, 0.65 mmol) to furnish ether 17e as a colorless oil (84 mg, 75%): rf = 0.8 (10% ea / hex); h nmr: 7.317.29 (m, 2h), 7.227.19 (m, 3h), 3.42 (t, j = 6.3 hz, 2h), 3.12 (s, 2h), 2.70 (t, j = 7.7 hz, 2h), 1.911.87 (m, 2h), 0.09 (s, 9h); c nmr: 142.3, 128.5, 128.2, 125.6, 74.2, 64.7, 32.3, 31.2, 2.98; hrms(tof - ms - ei+) calcd for c13h22osi (m): 222.1440; found: 222.1442; ir: 2955, 2845, 1246, 839 . [110458 - 41 - 8]: using the general procedure for etherification described above, peroxide 13b (115 mg, 0.5 mmol) was reacted with n - buli (0.34 ml 1.6 m in hex, 0.55 mmol) to furnish ether 17f as a colorless oil (73 mg, 78%). The h nmr data matched those previously reported: rf = 0.6 (5% ea / hex); c nmr: 75.3, 68.1, 36.7, 32.3, 31.8, 29.4, 25.6, 22.6, 19.7, 19.4, 14.0, 13.9 . [51182 - 98 - 0]: using the general procedure for etherification using a grignard reagent, peroxide 13b (115 mg, 0.50 mmol) was reacted with n - hexyl magnesium bromide (0.27 ml, 2 m in diethyl ether, 0.54 mmol) to furnish hexyl ether 17 g as a colorless oil (95 mg, 88%). The product has been partially characterized: rf = 0.6 (5% ea / hex); h nmr: 3.513.29 (m, 1h), 3.403.29 (m, 2h), 1.571.53 (m, 3h), 1.411.29 (m, 15h), 1.12 (d, j = 6.1 hz, 3h), 0.9 (m, 6h); c nmr: 75.3, 68.4, 36.7, 31.8, 31.7, 30.1, 29.4, 25.9, 25.6, 22.63, 22.62, 19.7, 14.05, 14.02 . [1927 - 68 - 0]: using the general procedure for etherification described above, peroxide 14b (101 mg, 0.50 mmol) was reacted with n - buli (0.31 ml 1.6 m in hexane, 0.5 mmol) to furnish ether 17h (12 mg, 15%) accompanied by 16 mg (15%) of recovered 14b . Partial spectral characterization has been reported.rf = 0.5 (15% ea / hex); h nmr: 4.59 (m, 1h), 3.89 (m, 1h), 3.76 (m, 1h), 3.52 (m, 1h), 3.40 (m, 1h), 1.84 (m, 1h), 1.72 (m, 1h), 1.651.49 (m, 6h), 1.451.35 (m, 2h), 0.94 (t, j = 7.3 hz, 3h); c nmr: 98.8, 67.3, 62.3, 31.8, 30.8, 25.5, 19.7, 1.4, 13.9 . [1927 - 63 - 5] using the general procedure for etherification with alkyl magnesium bromide described above, peroxide 14b (107 mg, 0.5 mmol) was reacted with n - hexyl magnesium bromide (0.25 ml, 2 m in diethyl ether, 0.50 mmol) to furnish hexyl ether 17i as a colorless oil (57 mg, 61%). The h nmr data matched those in a previous report.rf = 0.5 (15% ea / hex); h nmr: 4.57 (m, 1h), 3.903.84 (m, 1h), 3.763.70 (m, 1h), 3.523.47 (m, 1h), 3.413.35 (m, 1h), 1.871.79 (m, 1h), 1.741.68 (m, 1h), 1.621.50 (m, 6h), 1.421.26 (m, 6h), 0.89 (t, j = 6.8 hz, 3h); c nmr: 98.8, 67.6, 62.3, 31.7, 30.8, 29.7, 25.9, 25.5, 22.6, 19.6, 14.0 . Using the general procedure for etherification described above, peroxide 15b (107 mg, 0.50 mmol) was reacted with n - buli (0.34 ml, 1.6 m in hex, 0.55 mmol) to furnish ether 17j as a colorless oil (61 mg, 71%): rf = 0.70 (10% ea / hex); h nmr: 3.32 (t, j = 6.4 hz, 2h), 1.711.64 (m, 2h), 1.611.45 (m, 5h), 1.431.19 (m, 7h), 1.10 (s, 3h), 0.93 (t, j = 7.20 hz, 3h); c nmr: 72.8, 59.9, 36.5, 32.8, 25.86, 24.6, 22.2, 19.6, 13.99 . Hrms (tof - ms - ei) calcd for c11h22o: 170.1671; found: 170.1671; ir: 2962, 2925, 2862, 1447, 1081 . Using the general procedure for etherification described above, peroxide 15b (107 mg, 0.50 mmol) was reacted with n - hexyl magnesium bromide (0.27 ml, 2 m in ether, 0.54 mmol) to furnish hexyl ether 17k as a colorless oil (64 mg, 65%): rf = 0.65 (5% ea / hex); h nmr: 3.29 (t, j = 6.7 hz, 2h), 1.711.66 (m, 2h), 1.641.47 (m, 5h), 1.441.27 (m, 11h), 1.11 (s, 3h), 0.90 (t, j = 6.9 hz, 3h); c nmr: 72.9, 60.2, 36.5, 31.8, 30.7, 26.1, 25.8, 24.6, 22.6, 22.2, 14.0; hrms (tof - ms - ei) calcd for c13h26o: 198.1984; found: 198.1992; ir: 2962, 2925, 2862, 1447, 1081 . Using the general procedure for etherification described above, peroxide 16b (135 mg, 0.50 mmol) was reacted with n - buli (0.34 ml, 1.6 m in hex, 0.55 mmol) to furnish ether 17l as a colorless oil (86 mg, 76%): rf = 0.63 (10% ea / hex); h nmr: 5.735.66 (m, 1h), 5.595.52 (m, 1h), 4.01 (m, 0.23 h), 3.91 (dd, j = 0.6, 6.15, 1.76 h), 3.41 (t, j = 6.6 hz, 2h), 2.05 (m, 2h), 1.611.54 (m, 2h), 1.431.35 (m, 4h), 1.331.27 (m, 10h), 0.93 (t, j = 7.4 hz, 3h), 0.89 (t, j = 7.1 hz, 3h); c nmr: 134.5, 126.4, 71.6, 69.8, 32.3, 31.88, 31.87, 29.4, 29.27, 29.21, 29.0, 22.7, 19.3, 14.0, 13.9; hrms (tof - ms - ei): calcd for c15h30o: 226.2297; found: 226.2297; ir: 2954, 2923, 2852, 1465, 1105 . Using the general procedure for etherification described above, peroxide 16b (115 mg, 0.50 mmol) was reacted with n - hexyl magnesium bromide (0.27 ml, 2 m in ether, 0.54 mmol) to furnish hexyl ether 17 m as a colorless oil (104 mg, 81%): rf = 0.67 (10% ea / hex); h nmr: 5.735.66 (m, 1h), 5.595.52 (m, 1h), 4.01 (m, 0.24 h), 3.91 (dd, j = 0.6, 6.2, 1.76 h), 3.40 (t, j = 6.7, 2h), 2.05 (m, 2h), 1.58 (m, 2h), 1.431.27 (m, 18h), 0.900.87 (6h); c nmr: 134.5, 126.4, 71.6, 70.2, 32.3, 31.88, 31.74, 29.76, 29.46, 29.28, 29.22, 29.1, 25.9, 22.67, 22.62, 14.08, 14.03 . Hrms (tof - ms - ei) calcd for c17h34o: 254.2610; found: 254.2610; ir: 2954, 2923, 2852, 1465, 1105 . Reactions involving commercially available reagents were conducted as described above for sp c o bond formations except that reaction times were approximately 1 h. reactions involving in situ formation of vinyl or heteroaryllithium reagents from the corresponding tributylstannanes were conducted as described below . To a 78 c solution of alkenyl- or aryltributyltin (0.55 mmol) in dry thf (3 ml) was added n - buli (0.55 mmol, 1.6 m in hex). The reaction mixture was stirred for 10 min, after which was added a solution of the monoperoxyacetal (0.50 mmol) in thf (1 ml). The reaction mixture was stirred until no starting material could be detected on tlc (approximately 60 min) and then diluted with hex (10 ml). The resulting solution was passed though a column of neutral alumina (1 2 in .) And then concentrated (an aqueous workup could also be conducted employing aq . The residue was purified by silica flash chromatography using 1% ether / hex; chromatography of enol and thienyl ethers included 0.2% triethylamine . [51113 - 53 - 2]: using the general procedure described above, peroxide 8b (128 mg, 0.5 mmol) was reacted with phenyl lithium (0.30 ml, 1.8 m in dibutyl ether, 0.54 mmol) to furnish phenyl ether 18a as a colorless oil (102 mg, 87%). The same product was available (78 mg, 67%) by reaction of peroxide 8b (128 mg, 0.50 mmol) with phenyl magnesium bromide (0.18 ml, 3.0 m in ether, 0.54 mmol): rf = 0.66 (10% ea / hex); h nmr: 7.327.30 (m, 2h), 6.976.93 (m, 3h), 5.15 (m, 1h), 4.03 (m, 2h), 2.08 (m, 1h), 2.02 (m, 1h), 1.88 (m, 1h), 1.751.71 (m, 1h), 1.73 (s, 3h), 1.65 (s, 3h), 1.641.61 (m, 1h), 1.461.41 (m, 1h), 1.301.24 (m, 1h), 0.98 (d, j = 6.9 hz, 3h); c nmr: 151.1, 131.3, 129.4, 124.7, 120.5, 114.5, 66.1, 37.2, 36.1, 29.6, 25.8, 25.5, 19.6, 17.7 . Using the general procedure described above, peroxide 9b (118 mg, 0.5 mmol) was reacted with 2-methyl-1-propenyl magnesium bromide (1.3 ml, 0.5 m in thf, 0.65 mmol) to furnish ether 18b as a colorless oil (39 mg, 41%), which decomposed during chromatography: h nmr: 7.347.30 (m, 2h), 7.267.21 (m, 3h), 5.835.82 (m, 1h), 3.72 (t, j = 6.4 hz, 2h), 2.75 (t, j = 7.8 hz, 2h), 2.001.93 (m, 2h), 1.68 (s, 3h), 1.59 (s, 3h); c nmr: 141.8, 140.0, 128.5, 128.4, 125.8, 110.5, 70.7, 32.0, 31.4, 19.5, 15.0 . Hrms (tof - ms - ei) calcd for c13h18o (m): 190.1358; found: 190.1363; ir: 2918, 2865, 1689, 1496, 1159 . Using the procedure described above for etherifications with in situ generated sp rli, peroxyacetal 8b (128 mg, 0.5 mmol) was reacted with the reagent generated from tributyl vinylstannane (237 mg, 0.75 mmol) and n - buli (0.47 ml, 1.6 m in hex, 0.75 mmol) to furnish ether 18c as a colorless oil (62 mg, 66%), which partially decomposed upon thin - layer or column chromatography: rf = 0.7 (10% ea / hex); h nmr: 6.48 (dd, j = 6.8, 14.1 hz, 1h), 5.12 (t, j = 7.0 hz, 1h), 4.19 (d, j = 14.1 hz, 1h), 3.99 (d, j = 6.8 hz, 1h), 3.783.68 (m, 2h), 2.081.93 (m, 2h), 1.771.69 (m, 1h), 1.70 (s, 3h), 1.651.58 (m, 1h), 1.62 (s, 3h), 1.541.44 (m, 1h), 1.421.33 (m, 1h), 1.251.16 (m, 1h), 0.93 (d, j = 6.6 hz, 3h); c nmr: 152.0, 131.3, 124.7, 86.2, 66.3, 37.1, 35.9, 29.5, 25.7, 25.4, 19.5, 17.6 . Hrms (tof - ms - ei calcd for c12h22o (m h): 181.1598; found: 181.1602; ir: 2961, 2913, 2871, 1647, 1609, 1200 . Using the procedure described above for etherifications with in situ generated sp rli, peroxyacetal 8b (128 mg, 0.50 mmol) was reacted with the reagent generated from 2-(tributylstannyl)thiophene (279 mg, 0.75 mmol) and n - buli (0.47 ml, 1.6 m in hex, 0.75 mmol) to furnish ether 18d as a colorless oil (72 mg, 60%): rf = 0.75 (10% ea / hex); h nmr: 6.73 (dd, j = 3.7, 6.0 hz, 1h), 6.56 (dd, j = 1.2, 6.0 hz, 1h), 6.22 (dd, j = 1.2, 3.7 hz, 1h), 5.13 (t, j = 7.1 hz, 1h), 4.104.06 (m, 2h), 2.081.98 (m, 2h), 1.891.84 (m, 1h), 1.741.67 (m, 1h), 1.72 (s, 3h), 1.64 (s, 3h), 1.621.59 (m, 1h), 1.431.38 (m, 1h), 1.271.22 (m, 1h), 0.97 (d, j = 6.6 hz, 3h); c nmr: 165.8, 131.4, 124.7, 124.6, 111.7, 104.6, 72.3, 37.0, 36.0, 29.4, 25.7, 25.4, 19.5,17.7; hrms (tof - ms - ei) calcd for c14h22os (m): 238.1391; found: 238.1390; ir: 2963, 2925, 2913, 2871, 1536, 1193 . Using the procedure described above, peroxide 12b (129 mg, 0.50 mmol) was reacted with the reagent generated from 2-(tributylstannyl)thiophene (279 mg, 0.75 mmol) and n - buli (0.47 ml, 1.6 m in hex, 0.75 mmol) to furnish ether 18e as a colorless oil (65 mg, 54%): rf = 0.7 (10% ea / hex); h nmr: 6.746.72 (m, 1h), 6.566.54 (m, 1h), 6.236.21 (m, 1h), 4.04 (t, j = 6.4 hz, 2h), 1.831.76 (m, 2h), 1.481.44 (m, 2h), 1.381.30 (m, 12h), 0.91 (t, j = 6.8 hz, 3h); c nmr: 165.9, 124.6, 111.6, 104.5, 74.0, 31.9, 29.5, 29.3, 29.2, 25.8, 22.7, 14.1 . Hrms (tof - ms - ei) calcd for c14h24os (m): 240.1548; found: 240.1548; ir: 2920, 2853, 1536, 1456, 1193 . The difluoroether was prepared applying a previously reported two - step procedure (reaction of lithiated dithiane with the peroxide; fluorodesulfurization of the poorly stable difluoroether products) to three different precursors: 2-(3-phenylpropyl)-1,3-dithiane and monoperoxyacetal 10b (100 mg, 51% isolated yield); silyl peroxide 10d (105 mg, 54% isolated yield); t - butyl peroxide 10a (95 mg, 26%, with 35% recovered starting material). In each case, difluoroether 19 was isolated as a colorless oil with rf = 0.5 (5% ea / hex) and with spectra matching literature values . [123294 - 00 - 8] was prepared using a modification of a literature procedure . A solution of diisopropylamine (0.22 g, 1.1 equiv) in 5 ml of thf was cooled to 0 c, and n - buli (1.37 ml, 1.6 m in hex, 2.2 mmol, 1.1 equiv) was added . The reaction was stirred for 10 min, after which was added bu3snh (0.582 g, 2.0 mmol, 1 equiv). The reaction was stirred for 15 min at 0 c and then cooled to 78 c, whereupon hydrocinnamaldehyde (268 mg, 2.0 mmol) was added as a solution in thf (10 ml). After stirring at 78 c for 5 min, the separated organic layer was dried over mgso4 and concentrated (rotary evaporator, followed by high vacuum). The residue from the step described above was dissolved in dcm (12 ml), and the solution cooled to 10 c . Diisopropyl ethylamine (0.335 g, 1.3 equiv) was added, followed by chloromethyl methyl ether (0.177 g, 1.1 equiv). The reaction was stirred for 7 h at rt and then washed with water (10 ml). The separated organic layer was dried (na2so4) and concentrated on a rotary evaporator . The residue was chromatographed on silica gel using 1% ea / hex to afford 20 as a colorless liquid (63%, 598 mg): rf = 0.51 (10% ea / hex); h nmr: 7.337.27 (m, 2h), 7.237.19 (m, 3h), 4.65 (d, j = 6.5 hz, 1h), 4.62 (d, j = 6.5 hz, 1h), 4.154.11 (m, 1h), 3.41 (s, 3h), 2.822.66 (m, 2h), 2.222.05 (m, 2h), 1.601.49 (m, 6h), 1.391.29 (m, 6h), 0.960.88 (m, 12h), 0.92 (t, j = 7.3 hz, 3h); c nmr: 142.4, 128.4, 128.3, 125.7, 96.6, 73.7, 55.5, 37.4, 34.5, 29.2, 27.5, 13.7, 9.2 . To a 78 c solution of stannane 20 (235 mg, 0.5 mmol) in dry thf (3 ml) was added a solution of n - buli in hex (1.6 m, 0.5 mmol). After the reaction had stirred for 3 min, peroxide 11a (100 mg, 0.5 mmol) was added as a solution in thf (1 ml), and the reaction was stirred for an additional 30 min at 78 c . The cold bath was removed, and the reaction was allowed to warm to rt over 30 min . The reaction was quenched with water (1 ml) and diluted with hexane (40 ml). The mixture was washed with water (10 ml), and the separated aqueous layer was extracted with hex (10 ml). The residue was purified by silica gel chromatography using ether / hex (15%) to furnish recovered 11a (10%) and then the acetal 21a as a colorless oil (88 mg, 57%): rf = 0.36 (10% ea / hex); h nmr: 7.327.27 (m, 2h), 7.247.19 (m, 3h), 4.83 (d, j = 6.8 hz, 1h), 4.70 (d, j = 6.8 hz, 1h), 4.704.66 (m, 1h), 3.683.63 (m, 1h), 3.473.40 (m, 1h), 3.43 (s, 3h), 2.712.72 (m, 2h), 2.041.98 (m, 2h), 1.641.57 (m, 2h), 1.401.30 (m, 10h), 0.91 (t, j = 6.7 hz, 3h); c nmr: 141.7, 128.4, 125.8, 101.4, 93.3, 66.7, 55.7, 36.4, 31.8, 30.8, 29.8, 29.4, 29.2, 26.2, 22.6, 14.1; hrms (esi+ tof) calcd for c19h32o3 (m + na): 331.2249; found: 331.2265; ir: 2925, 2855, 1149, 1002 . The title compound was synthesized as a colorless oil (86 mg, 55%) from reaction of 9a (104 mg, 0.50 mmol) using the general procedure described above except that 1.2 equiv each of tributylstannane and n - buli was used: rf = 0.43 (10% ea / hex); h nmr: 7.357.31 (m, 4h), 7.287.21 (m, 6h), 4.85 (d,, j = 6.6 hz, 1h), 4.73 (d, j = 6.6 hz, 1h), 4.724.70 (m, 1h), 3.743.69 (m, 1h), 3.513.47 (m, 1h), 3.44 (s, 3h), 2.812.74 (m, 4h), 2.082.02 (m, 2h), 2.001.93 (m, 2h); c nmr: 141.88, 141.7, 128.5, 128.49, 128.45, 128.42, 128.39, 125.9, 125.8, 101.6, 93.5, 65.8, 55.8, 36.4, 32.4, 31.4, 30.8; hrms (tof ms es+) calcd for c20h26nao3 (m + na): 337.1780; found: 337.1775; ir: 2927, 1453, 1122, 1000, 697 . The title compound was synthesized as a colorless oil (89 mg, 53%) from stannane 20 (0.235 g, 0.50 mmol) and peroxyacetal 12b (0.129 g, 0.50 mmol) using the general procedure described above: rf = 0.40 (10% ea / hex); h nmr: 7.307.26 (m, 2h), 7.217.17 (m, 3h), 4.824.80 (m, 1h), 4.694.63 (m, 2h), 3.663.60 (m, 1h), 3.40 (s, 3h), 2.742.70 (m, 2h), 2.051.95 (m, 2h), 1.601.54 (m, 2h), 1.381.27 (m, 15h), 0.88 (t, j = 6.80 hz, 3h); c nmr: 141.9, 128.4, 125.9, 101.5, 93.4, 66.9, 55.8, 36.4, 32.0, 30.9, 29.9, 29.7, 29.6, 29.5, 29.4, 26.3, 22.7, 14.2 . Hrms (tof ms es+) calcd for c21h36nao3 (m + na): 359.2562; found: 359.2578; ir: 2925, 2856, 1149, 1000 . Using the general procedure described above, peroxide 8c (244 mg, 0.50 mmol) was reacted with alkoxylithium generated from reaction of stannane 20 (235 mg, 0.5 mmol) and n - buli (0.31 ml, 1.6 m in hex, 0.5 mmol) to furnish a mixture of mixed acetal 21d (48 mg, 28%) accompanied by 3-methoxymethyloxy-2-methyl-5-phenylpentan-2-ol 22 (23 mg, 20%). Nmr analysis of the crude reaction mixture suggested the presence of 40% of mixed acetal 21d; presumably, some was lost during the separation process . The mixed acetal 16d was contaminated with a small amount of inseparable byproduct, which could be removed by subjecting a dcm solution of the crude reaction mixture to brief (1 min) room temperature ozonolysis with 2% o3/o2 (1 mmol o3/min) prior to chromatography . The separated byproduct was determined to be 3-methoxymethoxypropyl benzene (3 mg, 3%), the product of protonation of the functionalized organolithium reagent . Rf = 0.5 (5% ea / hex); h nmr: 7.327.27 (m, 2h), 7.237.19 (m, 3h), 5.12 (m, 1h), 4.83 (d, j = 6.8 hz, 1h), 4.70 (d, j = 6.8 hz, 1h), 4.684.66 (m, 1h), 3.733.64 (m, 1h), 3.523.44 (m, 1h), 3.43 (s, 3h), 2.74 (m, 2h), 2.081.91 (m, 4h), 1.70 (s, 3h), 1.681.56 (m, 2h), 1.62 (s, 3h), 1.491.31 (m, 2h), 1.281.14 (m, 1h), 0.930.91 (m, 3h); c nmr: 141.7, 131.2, 128.4, 128.4, 128.39, 125.8, 124.7, 101.5, 101.4, 93.3, 64.9, 64.8, 55.7, 37.2, 37.1, 36.8, 36.78, 36.39, 30.83, 29.5, 25.7, 25.5, 19.6, 19.5, 17.6; hrms (tof - esi) calcd for c21h34o3na (m + na): 357.2406; found 357.2401; ir: 2927, 1454, 1369, 1002 . Rf = 0.3 (15% ea / hex); h nmr: 7.337.27 (m, 2h), 7.227.20 (m, 3h), 4.84 (d, j = 6.7 hz, 1h), 4.69 (d, j = 6.7 hz, 1h), 3.58 (s, 1h), 3.49 (s, 3h), 3.29 (m, 1h), 2.942.87 (m, 1h), 2.662.58 (m, 1h), 1.881.81 (m, 1h), 1.781.70 (m, 1h), 1.19 (s, 3h), 1.15 (s, 3h); c nmr: 141.9, 128.5, 128.4, 125.9, 99.2, 89.9, 72.0, 56.0, 33.5, 32.7, 26.3, 23.7 . Hrms (tof - esi) calcd for c14h22o3na (m + na): 261.1467; found: 261.1483; ir: 3461, 2930, 2889, 1028 . 3-methoxymethoxypropylbenzene [91898 - 11 - 2] has been reported on multiple occasions without nmr characterization: rf = 0.5 (20% ea / hex); h nmr: 7.337.28 (m, 2h), 7.237.18 (m, 3h), 4.66 (s, 3h), 3.57 (t, j = 6.4 hz, 2h), 3.39 (s, 3h), 2.73 (t, j = 7.8 hz, 2h), 1.991.89 (m, 2h); c nmr: 141.9, 128.4, 128.3, 125.8, 96.5, 67.1, 55.2, 32.4, 31.4 . The thp monoperoxyacetal (0.50 mmol) was dissolved in dry thf (5 ml), and the solution cooled to 0 c . Cyclopropyl magnesium bromide (1.3 ml, 0.5 m in thf, 0.65 mmol) was then added . The cooling bath was removed, and the reaction was stirred for 45 min prior to quenching by sequential dilution with 1 m aq . The combined hex extracts (20 ml 2) were dried over na2so4 and concentrated on a rotary evaporator . Diameter) using 12% ea / hex, with fraction concentrated initially at 80120 mm . (rotary evaporator, rt) and at 0.5 mm (1 min, 0 c). Using the general procedure described above, thp peroxide 8b (128 mg, 0.50 mmol) was reacted with cyclopropyl magnesium bromide (1.3 ml, 0.5 m in thf, 0.65 mmol) to furnish cyclopropyl ether 23a as a colorless oil (80 mg, 80%): rf = 0.52 (10% ea / hex); h nmr: 5.135.09 (m, 1h), 3.583.48 (m, 2h), 3.283.24 (m, 1h), 2.051.91 (m, 2h), 1.70 (s, 3h), 1.62 (s, 3h), 1.581.49 (m, 2h), 1.421.31 (m, 2h), 1.231.09 (m, 1h), 0.91 (d, j = 6.5 hz, 3h), 0.580.54 (m, 2h), 0.480.43 (m, 2h); c nmr: 131.1, 124.8, 68.9, 52.9, 37.2, 36.6, 29.5, 25.7, 25.4, 19.5, 17.6, 5.45, 5.41; hrms (tof - ms ci) calcd for c13h25o (m + h): 197.1905; found: 197.1913; ir: 2960, 2916, 1452, 1342 . Using the general procedure described above, thp peroxide 9b (118 mg, 0.50 mmol) was reacted with cyclopropyl magnesium bromide (1.3 ml, 0.5 m in thf, 0.65 mmol) to furnish cyclopropyl ether 23b as a colorless oil (64 mg, 72%): rf = 0.46 (10% ea / hex); h nmr: 7.327.29 (m, 2h), 7.227.19 (m, 3h), 3.52 (t, j = 6.4 hz, 2h), 3.28 (app septet, likely tt, j = 3.0, 6.0 hz, 1h), 2.70 (t, j = 7.7 hz, 2h), 1.941.87 (m, 2h), 0.610.57 (m, 2h), 0.490.45 (m, 2h); c nmr: 141.9, 128.4, 128.3, 125.8, 69.7, 53.0, 32.4, 31.2, 5.5; hrms: (tof - ms ei) calcd for c12h16o (m): 176.1201; found: 176.1199; ir: 3031, 2938, 2850, 1452, 1342 . Using the general procedure described above, thp peroxide 12b (129 mg, 0.50 mmol) was reacted with cyclopropyl magnesium bromide (1.3 ml, 0.5 m in thf, 0.65 mmol) to furnish cyclopropyl ether 23c as a colorless oil (79 mg, 80%): rf = 0.56 (10% ea / hex); h nmr: 3.49 (t, j = 6.7 hz, 2h), 3.26 (app septet, likely tt, j = 3.0, 6.0 hz, 1h), 1.581.54 (m, 2h), 1.341.27 (m, 14h), 0.89 (t, j = 7.0 hz, 3h), 0.570.55 (m, 2h), 0.471.95 (m, 2h); c nmr: 70.7, 52.9, 31.9, 29.68, 29.60, 29.57, 29.5, 29.3, 26.2, 22.7, 14.1, 5.4; hrms: (tof - ms ci) calcd for c13h27o (m + h): 199.2062; found: 199.2057; ir: 2922, 2853, 1453, 1343 . Using the general procedure described above, thp peroxide 13b (115 mg, 0.50 mmol) was reacted with cyclopropyl magnesium bromide (1.3 ml, 0.5 m in thf, 0.65 mmol) to furnish cyclopropyl ether 23d as a colorless oil (60 mg, 70%): rf = 0.6 (10% ea / hex); h nmr: 3.573.49 (m, 1h), 3.333.29 (m, 1h), 1.411.23 (m, 10h), 1.18 (d, j = 6.1 hz, 3h), 0.89 (t, j = 6.8 hz, 3h), 0.610.56 (m, 1h), 0.540.51 (m, 1h), 0.490.47 (m, 1h), 0.460.40 (m, 1h); c nmr: 75.7, 50.7, 36.6, 31.8, 29.4, 25.4, 22.6, 19.9, 14.0, 5.96, 5.42; hrms (tof - ms ei) calcd for c11h22o (m): 170.1671; found: 170.1678; ir: 2927, 2857, 1452, 1373 . [133476 - 12 - 7]: 2-bromo-2-methylpropyl benzene [23264 - 13 - 3] was prepared (3.79 g, 88%) as a colorless liquid from the corresponding alcohol (3 g, 20 mmol) using a modification of a reported procedure . Spectral data matched those in a previous report.rf = 0.8 (25% ea / hex); h nmr: 7.357.26 (m, 5h), 3.22 (s, 2h), 1.78 (s, 6h); c nmr: 137.3, 130.9, 128.0, 127.0, 66.6, 53.4, 33.9 . The t - buyl peroxide was prepared from the bromide (1.89 g 8.9 mmol, 1 equiv) through reaction with t - buooh (1.94 ml, 5.5 m in decane 1.2 equiv, 10.6 mmol), and silver trifluoroacetate (2.34 g, 10.6 mmol 1.2 equiv) using a reported procedure . Following a workup which includes brief exposure to nabh4 to destroy the trifluoroacetate ester, peroxide 24a was isolated as a colorless oil (0.79 g, 40%), which was only modestly responsive to the peroxide tlc stain (see the general experimental section) even after warming: rf = 0.7 (10% ea / hex). [830345 - 47]: 2-methyl-3-phenyl-1-propene (0.66 g, 5 mmol) and et3sih (1.16 g, 10 mmol) were sequentially added to a solution of co(acac)2 (0.50 mmol) in ethanol (15 ml). The reaction mixture was stirred under an atmosphere of o2 (balloon) until no starting material was observed (tlc, 14 h). The residue obtained upon concentration in vacuo was purified by silica gel chromatography using 18% ea / hex to furnish 24d (0.95 g, 68%) as a colorless oil . The molecule has previously been reported without nmr characterization: rf = 0.7 (10% ea / hex); h nmr: 7.287.18 (m, 5h), 2.88 (s, 2h), 1.16 (s, 6h), 1.00 (m, j = 7.9 hz, 9h), 0.70 (q, j = 7.9 hz, 6h); c nmr: 138.3, 130.8, 127.8, 126.1, 82.6, 44.8, 24.3, 6.9, 4.0 . To a 0 c solution of silyl peroxide 24d (0.70 g, 2.5 mmol) in thf (6 ml) was added n - bu4nf (3 ml, 1 m in thf, 3 mmol). After 10 min, the reaction was diluted with 50 ml of hex and the resulting solution was washed with water (2 5 ml). The dried (na2so4) organic layer was concentrated, and the residue was purified through a short plug of silica (10% ea / hexane) to furnish 2-hydroperoxy-2-methylpropyl)benzene [1944 - 83 - 8] as a colorless oil (0.41 g, 98%), which displayed spectral data matching literature reports.rf = 0.35 (10% ea / hex). The crude hydroperoxide (0.40 g, 2.4 mmol) and dihydropyran (0.2 g, 2.4 mmol) were reacted using method b to furnish monoperoxyacetal 24b as a colorless oil (0.397 g, 66%): rf = 0.42 (10% ea / hex); h nmr: 7.297.19 (5h), 5.12 (m, 1h), 4.06 (m, 1h), 3.63 (m, 1h), 2.97 (d, j = 13.5 hz, 1h), 2.86 (d, j = 13.5 hz, 1h), 1.79175 (m, 2h), 1.671.59 (m, 4h), 1.25 (s, 3h), 1.18 (s, 3h); c nmr: 138.0, 130.7, 127.9, 126.2, 101.2, 82.9, 62.8, 45.0, 27.9, 25.3, 24.7, 24.3, 20.1 . Hrms (tof - ms - es+) calcd for c15h22nao3 (m + na): 273.1461; found: 273.1461; ir: 2928, 2856, 1451 . Reaction of monoperoxyacetal24bwith organometallic reagents . Using the procedure described for reaction of n - buli with thp monoperoxyacetals, reaction of 24b (125 mg, 0.50 mmol) with n - buli (0.34 ml, 1.6 m in hex, 0.55 mmol) at 78 c for 15 min furnished, after workup and chromatography, recovered 24b (9 mg, 7%), alcohol 26 (15 mg, 10%), and butyl ether 25a (56 mg, 55%). Colorless oil; rf = 0.75 (10% ea / hex); h nmr: 7.297.26 (m, 2h), 7.237.19 (m, 3h), 3.42 (t, j = 6.6 hz, 2h), 2.79 (s, 2h), 1.591.52 (m, 2h), 1.441.35 (m, 2h), 1.14 (s, 6h), 0.94 (t, j = 7.3 hz, 3h); c nmr: 138.8, 130.7, 127.8, 126.0, 74.9, 61.2, 47.2, 32.8, 25.3, 19.6, 14.1; hrms (tof ms ei+) calcd for c14h22o (m): 206.1671; found: 206.1677; ir: 2956, 2927, 1453, 1362, 1080 . By the general procedure described for reaction of grignard reagents with monoperoxyacetals, a solution of 24c (125 mg, 0.5 mmol) in thf (3 ml) was reacted with hexylmgbr (0.60 ml, 2 m in ether, 2.4 equiv) to furnish, after workup and chromatography, hexyl ether 25b as a colorless oil (88 mg, 75%): rf = 0.75 (10% ea / hex); h nmr: 7.297.25 (m, 2h), 7.227.19 (m, 3h), 3.41 (t, j = 6.7 hz, 2h), 2.78 (s, 2h), 1.601.52 (m, 2h), 1.391.30 (m, 6h), 1.14 (s, 6h), 0.91 (t, j = 6.8 hz, 3h); c nmr: 138.8, 130.7, 127.8, 126.0, 74.9, 61.5, 47.1, 31.9, 30.7, 26.1, 25.3, 22.7, 14.2 . Hrms calcd for c16h26ona (m + na): 257.1879; found: 257.1881; ir: 2956, 2927, 1453, 1362, 1080 . Using the general procedure described for reaction of n - buli with thp peroxides as above, peroxide 24a (222 mg, 1.0 mmol) was reacted with n - buli (0.62 ml, 1.6 m in hex, 1.1 mmol) at room temperature for 5 h to furnish, following workup and chromatography, the following products: unreacted starting material (101 mg, 45%); alcohol 26 (48 mg, 32%); and pentylbenzene (5 mg, 3%): 2-methyl-1-phenylpropan-2-ol (26) [100 - 86 - 7]: colorless oil; rf = 0.35 (10% ea / hex); h nmr: 7.337.21 (m, 5h), 2.77 (s, 2h), 1.23 (s, 6h); c nmr: 137.9, 130.5, 128.3, 126.6, 70.8, 49.8, 29.2 . Pentyl benzene [538 - 68 - 1]: colorless oil; rf = 0.8 (10% ea / hex); h nmr: 7.317.29 (m, 2h), 7.237.19 (m, 3h), 2.63 (t, j = 7.7 hz, 2h), 1.671.62 (m, 2h), 1.401.33 (m, 4h), 0.93 (t, j = 7.0 hz, 3h); c nmr: 142.9, 128.4, 128.2, 125.5, 35.9, 31.5, 31.2, 22.5, 14.0 . Peroxide 24d (280 mg, 1.0 mmol) was reacted with n - buli (0.62 ml, 1.6 m in hex, 1 mmol) at room temperature for 6 h, to furnish a mixture of products which were separated by chromatography: recovered 24d (115 mg, 41%); et3sioh (15 mg, 24%); pentylbenzene (16 mg, 11%) and alcohol 26 (15 mg, 10%). Colorless oil; rf = 0.5 (10% ea / hex); h nmr: 1.491.44 (bs, 1h), 0.99 (t, j = 8.0 hz, 9 h), 0.618 (q, j = 8.0 hz, 6h); c nmr: 6.58, 5.79 . To a 78 c solution containing peroxyacetal 12b (0.50 mmol, 129 mg) and peroxide 9a (0.50 mmol, 104 mg) in thf (3 ml) was added n - buli (0.31 ml, 1.6 m in hex, 0.50 mmol). The reaction was quenched after 3 min with an excess of water to furnish, as a colorless oil, n - butyl decyl ether (27, 70 mg, 65%) accompanied by recovered 9a (90 mg, 86%). Butyl decyl ether (27) [111082 - 32 - 7]: rf = 0.8 (10% ea / hex); h nmr: 3.413.37 (m, 4h), 1.611.51 (m, 4h), 1.411.26 (m, 16h), 0.91 (t, j = 7.4 hz, 3h); 0.87 (t, j = 6.8 hz, 3h); c nmr: 71.1 . 70.7, 32.02 . 32.00, 29.9, 29.73, 29.69, 29.62, 29.4, 26.3, 22.8, 19.5, 14.2, 14.0 . Solutions of n - buli (0.47 ml, 0.762 mmol, 1.6 m in hex) and phli (0.42 ml, 0.762 mmol, 1.8 m in dibutyl ether, 0.42 ml) were sequentially drawn into the same 1 ml syringe, and the contents were rapidly added to a rapidly stirring 78 c solution of 9b (0.762 mmol, 180 mg) in dry thf (5 ml). The reaction was quenched at 78 c after 5 min to furnish dialkyl ether 28 (78 mg, 53%), phenyl alkyl ether 29 (30 mg, 19%), and biphenyl (5 mg, 4%): 3-butoxypropyl benzene (28): spectral data were nearly identical to those previously reported.rf = 0.7 (10% ea / hex); h nmr: 7.327.28 (m, 2h), 7.237.20 (m, 3h), 3.463.43 (m, 4h), 2.72 (t, j = 7.6 hz, 2h), 1.951.91 (m, 2h), 1.631.57 (m, 2h)1.441.40 (m, 2h), 0.96 (t, j = 7.4 hz, 3h); c nmr: 142.1, 128.5, 128.3, 125.7, 70.7, 69.9, 32.4, 31.9, 31.3, 19.4, 14.0 . 3-phenoxy propyl benzene [64806 - 63 - 9] (29): colorless oil; rf = 0.6 (10% ea / hex); h nmr: 7.347.21 (m, 7h); 6.996.92 (m, 3h); 4.00 (t, j = 6.3 hz, 2h), 2.85 (t, j = 7.5 hz, 2h), 2.18 (m, 2h); c nmr: 159.1, 141.7, 129.5, 128.6, 128.5, 126.0, 120.7, 114.7, 66.9, 32.3, 30.9 . Using the general procedure for etherification described above, methoxyethoxy peroxide 9e (105 mg, 0.5 mmol) was reacted with n - buli (0.34 ml, 1.6 m in hex, 0.55 mmol) to furnish 3-butoxypropyl benzene (28, 25 mg, 26%) as a colorless oil, accompanied by a small amount (4 mg, 6%) of 3-phenyl-1-propanol . Although no safety issues were encountered in the course of this work, any preparative work with peroxides should be conducted with an awareness of the potential for spontaneous and exothermic decomposition reactions . The reader is directed to a web - published collection of peroxide - related safety information.
Malignant apocrine lesions of the breast include apocrine ductal carcinoma in situ (adcis) and invasive apocrine carcinoma (iac) (1). Iac is a distinctive and rare subtype of breast malignancy that consists of more than 90% of apocrine differentiation (1, 2). The incidence of iac is reported as less than 1% to 4% of female invasive carcinoma (3). Microscopically, iac shows identical architectural growth pattern as invasive ductal carcinoma (idc) of no special type (nos), differing only in cytologic appearance (4). In addition, iac has a similar clinical presentation and overall patient survival rate similar to idc of nos (3). There are few case reports on radiologic findings of iac, but all cases show comparable malignant appearance to idc (5 - 8). Herein, we present a rare case of iac with its multiple radiologic modality findings that suggests a benign rather than malignant feature on initial ultrasonography (usg) and mammography and the pathologic correlation . A 61-year - old woman presented to our hospital due to an abnormal finding on screening mammography . There was no palpable lesion, pain or nipple discharge on physical examination at the time of the visit . The patient had a history of excision and biopsy on her left breast 9 years before that confirmed fibroadenoma in our medical center . Mammography showed a newly developed partly indistinct oval isodense nodule in the upper center of the right breast without any microcalcification (figure 1). Therefore, we categorized this lesion as breast imaging reporting and data system (bi - rads) category 0 and recommended breast usg . Usg images showed an oval circumscribed hypoechoic nodule about 1.0 cm in size with an internal cystic portion and peripheral vascularity in the upper center portion of the right breast that correlated with the mammographic finding (figure 2). Chest computed tomography (ct) showed a mild enhanced lesion in the upper center and outer portion of the right breast, but there was no evidence of lung metastasis (figure 3). Breast dynamic magnetic resonance imaging (mri) showed an approximately 1.1 cm sized oval irregular heterogeneous enhanced nodule in the right upper center portion . This nodule showed an early rim enhancement and delayed wash - out pattern (figure 4). Additional positron emission tomography (pet)/ct was performed and the nodule showed increased 18f - fluorodeoxyglucose (fdg) uptake of about 1.63 in maximum standardized uptake value (max suv) (figure 5). Pathologic study revealed abundant eosinophilic cytoplasm with prominent nuclei in the tumor (figure 6a). Immunohistochemical stain showed estrogen receptor (er), progesterone receptor (pr), human epidermal growth factor receptor 2 negativities, and gross cystic disease protein fluid-15 (gcdpf-15) positivity (figure 6b). This study was approved by the institutional review board of the soon chun hyang university cheonan hospital . It has been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . This study was approved by the institutional review board of the soon chun hyang university cheonan hospital . It has been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . The change of breast epithelium to apocrine epithelium is relatively common resulting in a wide range of benign and malignant pathologies (1). Malignant lesions due to apocrine change of the breast can be divided into adcis and iac (1). Iac was initially described by krompecher in 1916, and is now defined as more than 90% of apocrine differentiated neoplastic cells (1, 9). The incidence of iac is reportedly variable from less than 1% to 4% (8). However, the definite incidence of iac remains unknown currently, because there is no standardized consensus on the standard diagnostic criteria for iac, despite the diagnostic criteria of japaze et al . In 2005 iac is thought to have similar clinical, macroscopic and histological features as idc of nos (1, 3, 4). Both diseases have a greater likelihood in women over 40 (10). In general, hormonal factors such as nulliparity, first child after 30, early menarche, and late menopause increase the risk of breast cancer (1, 10). Continuous stimulation of sex hormones such as androgen seems to be related to the development of apocrine metaplasia in the breast (1). Physical examination varies from asymptomatic to the presence of a palpable mass with or without bloody nipple discharge (6, 10). The death rate of recurrent breast cancer occurs in 5 to 8% of the cases, which is similar to idc (10). Microscopically, iac presents almost the same architectural growth pattern as idc of nos, but it shows difference only in its cytological appearance (8). The cells are characterized by typical apocrine features such as abundant, eosinophilic granular cytoplasms, and prominent or multiple nucleoli (3). According to emerging evidence, apocrine carcinomas tend to show er, pr and bcl-2 negativity and androgen receptor positivity and expression of gcdpf-15 (1). A histopathological report indicated that gcdpf-15 positivity was correlated with small size, negative lymph node, and lower histologic grade of the tumor . Several reports suggest that apocrine carcinomas show a unique response to androgen (such as fluoxymesterone) administration as a part of treatment, but there is no standardized treatment option for iac (8). Radiologic appearance of iac is difficult to classify by the available modalities because of the limited information . Reported that most of the mammographic findings of iac are associated with microcalcification with no different features from idc (6). Also, onoue et al . Reported a case of iac that showed an oval circumscribed hyperdense nodule with microcalcification in mammography (7). Seo et al . Presented five case reports with mammographic findings in three cases (8). One case showed an irregular, partly indistinct hyperdense nodule and the other presented as an asymmetry (8). In contrast, our mammography showed a partly indistinct oval isodense nodule without any microcalcification . According to several previous case reports, iac shows variable sonographic appearances including irregular shaped, non - circumscribed solid mass with heterogeneous internal echo pattern (5, 7, 8). Reported two oval hypoechoic cysts with hypoechoic papillary projection with stromal invasion in some areas . Reported cases that showed two different iacs; one with a solid spiculated angular shape and the other as a complex cyst containing solid components and thick septa (5). Seo et al . Reported five cases that all showed irregular solid masses with heterogeneous internal echo patterns and noncircumscribed margins (8). In this case, usg showed an oval circumscribed hypoechoic nodule with an internal cystic change . To the best of our knowledge, there is only one case report on mri findings of iac . Seo et al . Reported iac with rapid, heterogeneous enhancement during the initial phase and washout in the delayed phase (8). Our mri showed oval spiculated nodule with rapid, rim enhancement and delayed washout pattern that was similar to the case reported by seo et al . Most authors agree that it is difficult to differentiate iac from idc by radiologic appearance alone (5 - 8). As described above, several previous case reports on radiologic findings of iac demonstrated relative malignant features; furthermore, it is accepted that it is not different from idc in radiologic appearance (5 - 8). In addition, it did not contain microcalcification in mammography, as in previous cases . To the best of our knowledge, this is the first case report on iac presenting as non typical malignant feature . In conclusion, radiologists should consider the malignant potential of newly developed nodule and evaluate the lesion pathologically despite the benign feature seen with radiologic imaging modalities.
The human brain changes itself in response to different types of experience through the reorganisation of its neuronal connections . It is suggested that it involves firstly a short - term modulation in the strength of existing pathways and that, over time, with prolonged exposure, such modulation might be followed by more stable, longer - term structural changes in brain networks . Neural plasticity manifests itself during brain development, motor and perceptual skill learning [3, 4], and also during / after central nervous system (cns) diseases / disorders, to name a few . The reorganisation of neuronal connections in the brain within these and also other contexts is commonly seen as being beneficial to the individual when brain changes are associated with improvements in the individual's behavioural capacity, neural plasticity is referred to as being adaptive [79]. On the other hand, when brain changes are linked to behavioural deterioration, or adverse consequences to the individual, neural plasticity is referred to as being maladaptive [10, 11]. Thus, it follows from the above that, by identifying neural plasticity and its behavioural correlates, together with an understanding of its mechanisms and likely causal factors, one can develop strategies to enhance adaptive and/or suppress maladaptive brain changes in order to improve the individual's behavioural capacity . This opportunity for intervention is of paramount importance to the clinical context of cns diseases / disorders, where many different patterns of neural plasticity have been identified, each of which being associated with either positive or negative behavioural outcomes . Motor deficits affecting the upper limb are a common manifestation of stroke and greatly contribute to decreasing the individual's functional performance and thereby to the level of disability that is achieved . It is widely appreciated that motor rehabilitation after stroke plays an essential role in reducing the individual's physical disability [1416]. This paper will focus on the motor rehabilitation of the paretic upper limb after stroke . Until about the late 1980s, neurorehabilitation professionals, despite recognising the importance of motor rehabilitation, had a somewhat restricted therapeutic armamentarium for treating stroke - related motor deficits, such as upper limb hemiparesis . This was in part due to our relatively limited understanding of the neural mechanisms underlying motor deficits / recovery after hemiparetic stroke at that time . By then, neurorehabilitation consisted mostly in teaching patients compensatory behaviours with their preserved body functions and/or in the utilisation of so - called neurophysiological approaches that lacked a strong scientific basis and had their therapeutic efficacy questioned . Fortunately, from that time onwards, with the advent of noninvasive neurophysiological and neuroimaging tools for assessing / altering brain activity / function in humans, and the increasingly greater utilisation of such tools in stroke patients, together with major advances in the development and use of animal models of stroke, the scenario began to change . For instance, over the past twenty five years or so, the findings from numerous correlational and experimental studies conducted with brain injured adult humans and animals have substantially enlightened our understanding of the neural plasticity that occurs after hemiparetic stroke [5, 2026]. Overall, this progress has contributed to the formulation of theories of motor recovery after stroke . In short, these theories identify neural plasticity patterns, both adaptive and maladaptive, and delineate their mechanisms and likely causal factors, for example, damage to, or activity changes in, particular brain regions or pathways and the presence or absence of specific behavioural or neural signals, to name a few . Such mechanistic understanding of poststroke motor deficits / recovery has, in turn, allowed for the theoretical conceptualisation and subsequent development of new, science - based motor rehabilitation strategies to treat upper limb hemiparesis, most of which are still under investigation [2730]. In parallel, other strategies that were being conceptualised and developed from otherwise different, yet complementary scientific perspectives, for example, behavioural psychology and multidisciplinary movement science, have found in those neural plasticity - based theories of motor recovery a strong neuroscientific support . This has further contributed to the establishment of these latter strategies as treatment options for poststroke upper limb hemiparesis [3134]. In this paper, we will refer to both these and those previously mentioned newly developed strategies as contemporary motor rehabilitation strategies . Altogether, this has not only represented a major revolution in neurorehabilitation but also nurtured a more optimistic prospect to the field . Notwithstanding the aforementioned achievements, several challenges have yet to be addressed in the arena of poststroke motor rehabilitation research, and this has consequences to clinical practice worldwide [3538]. One of these challenges, for instance, is that, in general, contemporary motor rehabilitation strategies for poststroke upper limb hemiparesis are most compatible with the recovery profiles of relatively high - functioning stroke survivors and therefore do not easily translate into benefit to individuals with poorer residual function . This is often in contrast to the day - to - day scenario of real - world rehabilitation settings, where many of the patients in need for motor rehabilitation after a stroke are usually at closer proximity to the lower end of the spectrum of functional recovery . This discrepancy may stem from the fact that most of the investigations performed so far in this field, particularly the studies testing motor rehabilitation strategies, have, for several reasons, focused primarily on well recovered stroke models (but see also). As a consequence, many stroke survivors, such as those sustaining low - functioning upper limb hemiparesis, remain with rather limited rehabilitation options . Therefore, in light of the above, we argue that there is a need for alternative motor rehabilitation strategies for stroke, if clinical practice demands are to be met more widely and effectively . First, they should be able to promote the adaptive neural plasticity pattern(s) currently identified as instrumental to poststroke motor recovery . Second, and most critically, their ability to do that must not be influenced by the individual's level of residual function . Importantly, such strategies are likely not only to be more compatible with the recovery profiles of stroke survivors with poor residual function, but also to translate into benefit to the entire spectrum of functional recovery . However, in order for this to be achieved, models that more closely resemble a condition of low - functioning upper limb hemiparesis are required . As will be elaborated in the remainder of this paper, upper limb immobilisation is a key candidate for this position . Below, we will first briefly describe two prevailing theories of motor recovery after stroke and some of the contemporary motor rehabilitation strategies for treating poststroke upper limb hemiparesis that have been largely underpinned by these theories . This will be followed by an overview of upper limb immobilisation studies in healthy adult humans and animals and a discussion as to how the findings from these studies could inspire the creation of a model that, we believe, is likely to be of particular relevance to the context of motor rehabilitation after stroke . Our premise here is that an upper limb immobilisation model, by capitalising on both, current theories of motor recovery after stroke and the shortage of physical or overt movements, which is a hallmark of low - functioning hemiparesis, offers a compelling neurobehavioural framework upon which alternative motor rehabilitation strategies for treating upper limb hemiparesis can be envisioned, firstly developed and tested in healthy individuals, and then ultimately translated into the clinical context of poststroke motor rehabilitation . The motor cortex contains representations of body parts [42, 43]. Throughout this paper these representations are commonly derived through electrical stimulation of the precentral cortex in the frontal lobe, with either noninvasive or invasive techniques, such as transcranial magnetic stimulation (tms) and intracortical microstimulation (icms), respectively . While tms is more frequently used with humans, icms is typically employed in animal research . The mechanism of action of these two techniques, when they are used for that purpose, usually involves the stimulation of axons from local intracortical circuits that synapse onto corticospinal neurons that then synapse onto contralateral spinal motor neurons innervating skeletal muscles, which, in turn, act upon specific body parts . However, direct activation of corticospinal neurons may also occur under certain conditions with both tms and icms . The response to stimulation is then recorded in the periphery visually, that is, through the visualisation of movement of the corresponding body part(s), and/or by means of surface electromyography of the involved muscle(s). Thus, when derived, cortical motor representations may be considered as being a measure of both the amount of cortical / corticospinal tissue that is being dedicated to the motor control of a particular body part, which can be inferred from the size of the obtained representation over the cortex or scalp, and the strength or efficacy of this control at the time of stimulation, which can be inferred from the intensity of the recorded response in the periphery . The latter, in turn, usually indicates the excitability of the cortical / corticospinal components of the representation that are activated by the stimulation . As phillips and porter (1977) commented on the use of electrical stimulation for deriving cortical motor representations, this leaves us free to concentrate on its merits as a tool for mapping the outputs that are available for selection by the intracortical activities that it cannot itself evoke (phillips and porter 1977, p. 37) (, p. 304). In other words, the size and/or excitability of a cortical motor representation corresponding to a particular body part can be thought of as reflecting the individual's motor capacity / skill with that body part . Contrary to what was once held, cortical motor representations are by no means static entities . Instead, numerous neurophysiological studies performed with tms and icms on adult humans and animals in the past years have consistently demonstrated that such representations are rather flexible or dynamic and that one fundamental driver of their organisation, in terms of both their size and excitability, is the amount of use or sensorimotor experience with the corresponding body part(s). In general, conditions of increased use or sensorimotor experience that increase activity in the efferent and/or afferent neural signalling pathways targeting and/or coming from a particular body part, or parts, for example, motor skill learning / acquisition and somatosensory stimulation, induce an increase in the size and/or excitability of the cortical motor representation(s) of the involved body part(s). This is often accompanied by gains in motor capacity with the involved body part(s) and therefore reflects adaptive neural plasticity . Conversely, conditions of decreased use or sensorimotor experience that decrease or even cease activity in those pathways, for example, brain or peripheral nerve lesions, amputation, spinal cord injury, and ischemic nerve block - mediated local anesthesia, lead to a decrease in the size and/or excitability of the cortical motor representation(s) of the affected body part(s). This usually parallels a reduction in motor capacity with the affected body part(s) and therefore reflects maladaptive neural plasticity [4649]. There is evidence that these changes in the size and/or excitability of cortical motor representations, both adaptive and maladaptive, are mediated by, among other factors, synaptic strength modification processes, such as long - term potentiation (ltp) and long - term depression (ltd), occurring within intracortical circuits in the motor cortex (see [45, 49] for further details). Findings collated from several neurophysiological and neuroimaging investigations performed with brain injured adult humans and animals have contributed so far to the formulation of at least two complementary theories of motor recovery after hemiparetic stroke . In the following sections, we will refer to these theories as the reactivation and rebalancing theories . It might be worth mentioning that what we call theories here has actually appeared more frequently in the literature as concepts, models, or neural strategies for motor recovery, rather than as theories per se . Also, other names rather than nevertheless, the underlying principles / mechanisms have been fully preserved and the terms employed here were chosen simply for the purposes of this paper . First, in the healthy brain, increased use or sensorimotor experience in the form of motor skill acquisition promotes adaptive neural plasticity, that is, increases in the size and/or excitability, of the cortical motor representation(s) of the involved body part(s). Second, motor deficits after hemiparetic stroke are due not only to the structural lesion itself, but, critically, also to maladaptive neural plasticity occurring in structurally intact, residual brain areas connected to the damaged region(s). After the stroke, this region may still contain some residual cortical / corticospinal components of the cortical motor representations corresponding to the paretic body parts . When this is the case, it follows that, over time, these spared representations often undergo a substantial reduction in their size and/or excitability . This condition of perilesional depression is usually the combined result of phenomena that are initially triggered by the stroke lesion, such as diaschisis and learned nonuse, and that are subsequently aggravated by a state of substantially reduced usage of, and hence reduced sensorimotor experience with, the paretic body parts . Third, the mechanisms of the above can interact so that after hemiparetic stroke, increased use or sensorimotor experience with the paretic body parts in the form of motor skill (re)acquisition may adaptively modulate perilesional neural plasticity . Therefore, this theory predicts that, by increasing use of the paretic body parts in a skill (re)acquisition - like manner, the cortical motor representations corresponding to these body parts, which may still have some residual components available in the perilesional tissue, are reactivated and adaptively stimulated; that is, they increase their size and/or excitability and thereby improve the individual's motor function [50, 51] (see also [20, 52, 53] for further discussion). Theory, on the other hand, suggests that motor deficits after hemiparetic stroke result, apart from the structural lesion itself, from an interaction between depression of the perilesional tissue and further maladaptive neural plasticity involving both the ipsilesional and the contralesional cerebral hemisphere . For instance, a hemiparetic stroke, besides selectively disrupting lateralized motor control networks, often compromises transcallosal circuits that regulate the interhemispheric interactions between cortical areas . Of particular relevance here are the inhibitory transcallosal circuits that connect the cortical motor representations in the motor cortex of one hemisphere to those in the motor cortex of the contralateral hemisphere . The result is a reduction in the inhibition from the motor cortex of the stroke - affected hemisphere to the homologous area in the opposite, unaffected hemisphere . Such disinhibition usually leads to an abnormally increased activity in the contralesional motor cortex, which, in turn, causes excessive transcallosal inhibition from this area towards the homologous region in the ipsilesional hemisphere . This phenomenon is likely to be aggravated by a concomitant compensatory increased use of the less - affected body side, which contributes to further increasing activity in the contralesional motor cortex . Overall, it is suggested that such abnormal interhemispheric inhibition would be superimposed to an already existing condition of reduced perilesional activation in the affected hemisphere and that this could, in turn, contribute to a further decrease in the size and/or excitability of potentially available residual cortical motor representations in that hemisphere . Accordingly, this theory predicts that motor recovery after hemiparetic stroke is facilitated / enhanced if the interhemispheric interactions between the cortical motor representations of the two homologous motor cortices are rebalanced and that this can be achieved by increasing activity in the ipsilesional and/or decreasing activity in the contralesional motor cortex [5456] (see also [20, 21, 57] for further discussion). Overall, those two aforementioned theories of motor recovery currently form the neuroscientific basis of contemporary motor rehabilitation strategies for treating upper limb hemiparesis after stroke . These strategies might be broadly grouped into two complementary categories of interventions . The first category is primarily supported by the reactivation theory and currently constitutes the central pillar of modern neurorehabilitation . This category aims to increase skilled use of the paretic upper limb in order to reactivate and adaptively stimulate, that is, increase the size and/or excitability of, likely latent cortical motor representations in the damaged cerebral hemisphere . It consists mostly of physiotherapy exercises that are performed with the paretic upper limb and that are delivered in the form of repetitive and increasingly challenging task - specific exercises directed towards the (re)acquisition of motor skills [58, 59]. The second category of interventions, on the other hand, is based on both theories of motor recovery, but particularly on the rebalancing theory . This category aims to boost the effects of physiotherapy exercises through the use of adjunctive therapies that have the potential to rebalance the interhemispheric interactions between the two homologous motor cortices and that can be delivered in combination with task - specific practice . These adjunctive therapies include, but are not limited to, excitatory and inhibitory brain stimulation (+ bs and bs, resp .) [6070] and peripheral somatosensory stimulation (pss) [7175]. The standard approach here is to deliver + bs to the ipsilesional motor cortex to increase its activity and/or bs to the contralesional motor cortex to decrease its activity . In the case of pss, the stimuli, which typically consist of low - intensity electric currents, are delivered transcutaneously to the paretic body part(s) in order to increase activity of the contralateral, ipsilesional motor cortex . For instance, task - specific exercises can be coupled with both bs and pss in order to further potentiate practice - induced adaptive neural plasticity (e.g., see). Despite their promise, it is worth noting that these, and also other adjunctive therapies, are still under investigation and therefore are not yet widely used in clinical practice . The two categories of interventions are illustrated in figure 1(b). As can be noted, contemporary motor rehabilitation strategies for treating poststroke upper limb hemiparesis individuals are required to have a level of residual function that will ultimately allow them not only to actively engage with repetitive task practice, but also to perform increasingly difficult exercises [58, 59]. Therefore, while these rehabilitation strategies may undoubtedly translate into benefit for some stroke survivors, their overreliance on the availability of relatively high levels of residual function to overcome maladaptive and/or promote adaptive neural plasticity, that is, to promote motor recovery, represents a major obstacle . This is particularly true for individuals sustaining low - functioning upper limb hemiparesis who, due to poor residual function, cannot effectively engage with the physical practice of motor tasks in the way that is needed to promote the adaptive neural plasticity driving functional improvements . For these individuals,, it is worth clarifying that a condition of poor residual function after a hemiparetic stroke, that is, low - functioning hemiparesis, at least in the way this concept is used in this paper, does not necessarily imply a severe neurological lesion completely destroying the cortical and/or corticospinal components of the cortical motor representations corresponding to the paretic body parts . While it stands to reason that such a severe lesion would result in poor residual function, the occurrence of the former is not a sine qua non for the presence of the latter . This is because many factors can influence / determine the level of residual function that is expressed by an individual after a hemiparetic stroke . For instance, it is well recognised that patients often experience substantial cardiorespiratory and skeletal muscle deconditioning after a hemiparetic stroke [77, 78]. As we have discussed elsewhere (e.g., see), such deconditioning status, in turn, might greatly contribute to increasing their fatigue levels . Altogether, this can critically decrease the individual's physical capacity and ability to actively engage with the repetitive overt practice of progressive motor tasks, hence contributing to a low - functioning profile . Moreover, as already mentioned in the previous section, the motor deficits and hence the level of residual function that is shown by an individual after a hemiparetic stroke are currently thought to be largely influenced by maladaptive neural plasticity patterns occurring in structurally intact, residual brain areas / networks that were otherwise spared by the lesion . Therefore, a more holistic view here would be that a condition of low - functioning hemiparesis after stroke likely results from a complex interaction among different compromised body systems, instead of simply from the more direct effects of the neural damage, that is, the selective destruction of motor control pathways in the brain . Thus, as suggested earlier in this paper, alternative motor rehabilitation strategies for stroke are currently needed . Essentially, in order to overcome the present obstacle, these alternative strategies must not rely on the individual's level of residual function to move from a state of maladaptive to one of adaptive neural plasticity, which may eventually translate into improved motor function . As will be discussed henceforth, an upper limb immobilisation model might be an attractive framework for developing such strategies . Given the fundamental role of use or sensorimotor experience in shaping cortical motor representations and thereby the individual's motor capacity / skill with the corresponding body part(s) (see section 2.1), studies have started to investigate upper limb immobilisation as a paradigm of disuse or sensorimotor deprivation . In such studies, the entire upper limb, or part of it, is prevented to move by means of a bandage, splint, cast, and/or sling, either because of trauma or simply for experimental purposes . Particularly, these investigations have focused not only on the neural but also on the behavioural effects of immobilisation . (2006) immobilised the left arm and hand of healthy participants for 12 consecutive hours to explore the effects of sensorimotor deprivation on sensorimotor cortex activity, motor performance, and sleep slow wave activity (swa). After the immobilisation, the authors found a substantial decrease in neuronal activity in the hand representation of the right sensory and motor cortices, as revealed by reduced somatosensory and motor potentials evoked through peripheral nerve stimulation and tms, respectively . At the same time, the motor performance of the immobilised arm and hand had deteriorated, as indicated by an increase in hand - path area variability while individuals were reaching toward targets placed in front of them . Also, after immobilisation, there was a localised decrease in sleep swa over the right sensorimotor cortex, which was detected through electroencephalography during subsequent sleep . Of note, a positive correlation was found between the changes in motor performance and the changes in both somatosensory evoked potentials and sleep swa . Subsequent studies have revealed comparable findings regarding the behavioural effects of immobilisation . In one of these studies, healthy participants had their upper limb immobilised for either 6 or 12 consecutive hours . It was found that after 12 but not 6 hours of immobilisation, motor control of the restricted limb was impaired, which was expressed through abnormalities in both hand trajectories and inter - joint coordination during reaching movements . Similarly, in a different study, healthy individuals displayed altered motor performance in a reach - to - grasp task after 10 hours of continuous arm and hand immobilisation . Here, the transport phase of the reach - to - grasp movement was affected, in a way that reaching was slower and its peak velocity time was achieved earlier . An interesting finding from this study was that motor performance on the reach - to - grasp task quickly returned to baseline levels with only a few trials of practice after the immobilisation had been removed . By employing the same immobilisation protocol from the latter study above, avanzino et al . (2011) explored with tms the effect of upper limb disuse on the interhemispheric interactions between the two homologous motor cortices . Additionally, they investigated whether this effect was modulated by the amount of use of the nonimmobilised limb . The study consisted of two groups . In one group, participants received no instructions regarding the amount of use of the nonimmobilised arm and hand, free to move group, whereas, in the other group, volunteers were instructed to limit contralateral movements, limited movement group . After the 10 consecutive hours of right arm and hand immobilisation, both groups showed decreased excitability of the hand representation in the left motor cortex and reduced interhemispheric inhibition from the left to the right hand cortical motor representation, with the latter effect being more pronounced in the free to move group . Of note, the excitability of the hand representation in the right motor cortex, as well as the interhemispheric inhibition from the right to the left hand cortical motor representation, increased only in the group that was free to move the left, nonimmobilised arm and hand . In keeping with these latter findings, a longitudinal neuroimaging study by langer et al . (2012) reported bilateral structural changes in the sensorimotor cortex and corticospinal tract of immobilised individuals recovering from upper limb fractures . After an average of 16 days of right arm and hand immobilisation, cortical thickness and fractional anisotropy (fa) were reduced in the hand representation of the left sensory and motor cortices and over the left corticospinal tract, respectively . In addition, motor abilities with the left arm and hand improved throughout the restriction period, presumably as a consequence of an increased use in order to compensate for the immobilisation of the contralateral limb . Interestingly, this behavioural change was associated with an increase in both cortical thickness and fa of the right motor cortex and corticospinal tract, respectively, and with a decrease in cortical thickness over the left sensorimotor cortex . In another longitudinal study, in this case conducted with healthy adult monkeys, milliken and colleagues (2013) investigated the effect of distal forelimb immobilisation on the somatotopic organisation of the corresponding cortical motor representation . Here, immobilisation periods varied from 38 to 248 days . Detailed cortical motor representations of the distal forelimb of the animals were obtained through icms techniques before, during, and after the immobilisation intervals . The authors found a progressive decrease in the size of the representation of the digits together with an equivalent increase in the size of the representation of more proximal limb parts, such as wrist and forearm . These changes were paralleled by a reduction in general / skilled use of the digits, which, in turn, was followed by a concomitant increase in the use of more proximal limb parts . In general, those cortical changes were reversed to baseline levels after removal of the immobilisation during a period of behavioural recovery, when the animals regained general / skilled use of the digits, either spontaneously or through forced use . Complementing the above findings, rosenkranz et al . (2014), by recording a variety of tms - derived measures, recently showed that upper limb immobilisation, besides decreasing the excitability and/or size of the corresponding cortical motor representations in the contralateral motor cortex, also selectively alters neural plasticity and intracortical inhibition mechanisms within the same representations . Specifically, the authors showed that, after 8 consecutive hours of left hand immobilisation, individuals displayed increased responsiveness to paired associative stimulation (pas) protocols, which are thought to induce long - term potentiation- (ltp-) like and long - term depression- (ltd-) like processes . In other words, both ltp and ltd were facilitated in the hand representation of the contralateral right motor cortex after immobilisation, presumably reflecting homeostatic adjustments that operate to increase the sensitivity of corticospinal neurons to available synaptic inputs and thereby prevent too much of a decrease in motor output capacity . In addition, after immobilisation, short - interval intracortical inhibition (sici) was reduced in the hand representation of the right motor cortex, and this change was negatively correlated with the changes in excitability over this cortical area . Again, this is likely to reflect compensatory adjustments that act during / after immobilisation to maintain overall motor cortex excitability within a certain range . Finally, a correlation was also found between the reduction in the excitability of the hand representation over the right motor cortex and the strength of the effect of the pas protocols . This correlation was positive for the ltp - inducing and negative for the ltd - inducing protocol . The findings from this study, particularly those regarding the changes in neural plasticity and intracortical inhibition mechanisms, might explain, at least in part, the somatotopic reorganisation of cortical motor representations that usually occurs with upper limb immobilisation, such as that observed in the monkeys from the study mentioned previously . Interestingly, the neural and behavioural effects that have been reported in immobilisation studies are, to a large extent, similar to those effects reported in investigations employing ischemic nerve block - mediated local anaesthesia, both in healthy individuals and after stroke [8790]. This similarity emphasises the role of the presence / level of activity in the afferent and efferent neural signalling pathways associated with a particular body part in determining the size and/or excitability of its representation in the motor cortex and thereby the individual's motor capacity / skill with it . This observation, in turn, further corroborates the reactivation theory that was described previously in this paper (see section 2.2). In summary, the studies reviewed in this section provide evidence that upper limb immobilisationimpairs motor performance with the restricted body part(s), even after short periods of restriction ranging from 10 to 12 hours, an effect that is largely reversible;induces maladaptive neural plasticity of the cortical motor representation(s) of the immobilised body part(s), which expresses itself in the form of motor behaviour deterioration, and which may vary from a simple reduction in the size and/or excitability of the involved representation(s) to structural changes in the cortical and corticospinal components of the same representation(s), depending on the duration of the immobilisation;affects the interhemispheric balance between the two homologous motor cortices in favour of the cortical motor representation(s) from the cerebral hemisphere ipsilateral to the immobilised body part(s), an effect that seems to be largely modulated by the amount of use of the contralateral, nonimmobilised limb;modulates neural plasticity and intracortical inhibition mechanisms within the cortical motor representation(s) of the restricted body part(s), a process which is likely to be responsible for the maladaptive neural plasticity of the involved representation(s). More specifically, such modulation occurs in a direction that appears to be predicted by the amount of depression that is induced by the restriction in the involved representation(s); for example, the larger the decrease in excitability, the greater the sensitivity of the representation(s) to subsequent ltp - like processes (with the opposite being true for ltd - like processes) and the smaller the reduction in sici within the same representation(s). Impairs motor performance with the restricted body part(s), even after short periods of restriction ranging from 10 to 12 hours, an effect that is largely reversible; induces maladaptive neural plasticity of the cortical motor representation(s) of the immobilised body part(s), which expresses itself in the form of motor behaviour deterioration, and which may vary from a simple reduction in the size and/or excitability of the involved representation(s) to structural changes in the cortical and corticospinal components of the same representation(s), depending on the duration of the immobilisation; affects the interhemispheric balance between the two homologous motor cortices in favour of the cortical motor representation(s) from the cerebral hemisphere ipsilateral to the immobilised body part(s), an effect that seems to be largely modulated by the amount of use of the contralateral, nonimmobilised limb; modulates neural plasticity and intracortical inhibition mechanisms within the cortical motor representation(s) of the restricted body part(s), a process which is likely to be responsible for the maladaptive neural plasticity of the involved representation(s). More specifically, such modulation occurs in a direction that appears to be predicted by the amount of depression that is induced by the restriction in the involved representation(s); for example, the larger the decrease in excitability, the greater the sensitivity of the representation(s) to subsequent ltp - like processes (with the opposite being true for ltd - like processes) and the smaller the reduction in sici within the same representation(s). The proposal presented in this paper is that an upper limb immobilisation model offers a compelling neurobehavioural framework for developing alternative motor rehabilitation strategies for treating upper limb hemiparesis after stroke . As summarized previously, the neural effects of upper limb immobilisation are, to a large extent, similar to the maladaptive neural plasticity patterns that are often seen after hemiparetic stroke and that underpin current theories of motor recovery . To briefly recall, those effects involve both a reduction in the size and/or excitability of the cortical motor representation(s) corresponding to the immobilised body part(s) and changes in interhemispheric balance, with increased activity biased towards the cortical motor representation(s) from the cerebral hemisphere ipsilateral to the restricted limb . Indeed, in terms of maladaptive neural plasticity patterns, an upper limb immobilisation model largely resembles a hemiparetic stroke model, except of course for the absence of a true lesion (figure 2(a)). But if this is to be true, then what could be the advantages of using an upper limb immobilisation model as a stroke - like model for developing alternative motor rehabilitation strategies for upper limb hemiparesis, in place of currently used models (see section 1.1)? Here, we argue that, besides inducing the maladaptive neural plasticity patterns that usually occur after a hemiparetic stroke and that are currently thought to play an important role in mediating the individual's motor deficits, the upper limb immobilisation model essentially promotes a condition of rather decreased, if any, overt use or sensorimotor experience, which in fact characterizes the restriction paradigms themselves . Such condition, in turn, is much more compatible with the recovery profiles of individuals with poor residual function . Thus, an upper limb immobilisation - based stroke - like model, in comparison to current models, more closely resembles a condition of low - functioning upper limb hemiparesis . Importantly, within this context, if during a paradigm of upper limb immobilisation in healthy individuals interventions could be delivered in order to prevent the maladaptive neural plasticity that is induced by movement restriction, because these interventions would necessarily have to bypass overt movement execution, they could translate into alternative motor rehabilitation strategies for poststroke upper limb hemiparesis with a greater potential for benefiting those with poor residual function . Here, it is at least theoretically plausible that such interventions would be able to promote, for example, in low - functioning stroke survivors, the adaptive neural plasticity patterns currently identified as instrumental to poststroke motor recovery (see the reactivation and rebalancing theories in section 2.2). Furthermore, the behavioural effects of immobilisation would also be of value to this context . For instance, it would be the case that interventions under investigation could aim to prevent not only the induction of maladaptive neural plasticity but also the deterioration of overt motor performance, which has also been a reported effect of immobilisation early after its removal (see beginning of section 4). Besides focusing on the neural and behavioural effects of upper limb immobilisation, recent studies have also started to explore how it can be used as a model of maladaptive neural plasticity upon which it is possible to test interventions that aim to prevent this neural plasticity from occurring . Specifically, researchers have employed upper limb immobilisation firstly to induce a depression, that is, a decrease in the size and/or excitability, of the cortical motor representation(s) of the restricted body part(s) and/or an interhemispheric imbalance between homologous representations in favour of the representation(s) ipsilateral to the immobilised body part(s). Then, they would try to prevent these changes from occurring through the concomitant delivery of interventions that are thought to have the potential to activate and adaptively stimulate cortical motor representations in the absence of overt use or sensorimotor experience with the corresponding body part(s). Below, we will review these studies and interventions within the context of motor rehabilitation after stroke . They include, for example, motor imagery (mi) and action observation (ao). These strategies are referred to as covert motor strategies because of their intrinsic ability to activate the motor system of the brain without the overt execution of movements . As suggested by sharma et al . (2006), they may achieve that through the backdoor of the brain's motor system . Theoretical support for mi and ao as covert motor strategies comes mostly from the simulation theory, which was proposed by jeannerod almost fifteen years ago . According to this theory, the brain's motor system, including its cortical motor representations in the motor cortex, is part of a simulation network that is activated not only when we move, but also when we imagine ourselves or observe others moving . In this vein, the theory proposes that the neural substrate that is activated for the overt execution of a movement or action is, to a large extent, also activated by the imagination or observation of that same movement or action . Studies from our group and several others performed with healthy participants have provided strong empirical support for the simulation theory, by showing an extensive overlap in neural activation between conditions of overt execution and conditions of imagination or observation of movements, and this includes activation of the cortical motor representation(s) of the involved body part(s) [9398]. In addition, we have also shown that this overlap in neural activity does not remain confined to the motor execution domain but also occurs, for example, between movement preparation and mi [99, 100]. This equivalence in neural activation between movement preparation / execution and mi / ao is thought to account for the improvements in overt motor performance that are commonly seen in healthy individuals after mi / ao training [94, 101, 102]. For example, in a classical study, pascual - leone and colleagues (1995) showed that mi training alone, in comparison to physical practice, also led to significant improvements in the overt performance of a fine motor skill with the hand . Moreover, the same neural effects that were induced by physical practice were also found in the individuals undergoing only mi training . In both conditions, there was an increase in the size and excitability of the cortical motor representation of the trained hand . Importantly, the findings from this study demonstrate not only that mi on its own can activate the cortical motor representation(s) of the involved body part(s) but, more critically, that mi training can lead to adaptive stimulation of such representation(s). This adaptive stimulation, in turn, translates into improved overt motor performance with the involved body part(s). Overall, the above findings have spurred the infusion of mi and ao into neurorehabilitation, particularly into the context of poststroke motor rehabilitation [103105]. Essentially, the underlying assumption here is that mi or ao training can reactivate and adaptively stimulate potentially spared cortical motor representations in the stroke - affected hemisphere . This, in turn, would contribute to increasing activity in the ipsilesional motor cortex and thereby rebalance the interhemispheric interactions between this cortical area and the homologous region in the contralateral, unaffected hemisphere . Collectively, this would contribute to improvement of the individual's motor function [91, 106108] (see the reactivation and rebalancing theories in section 2.2). Interestingly, studies by our group and others have shown that the aforementioned similarity reported in healthy individuals between overt movement and mi / ao, in terms of both the neural substrate that is activated and the ability of both overt movement and mi / ao to induce improvements in overt motor performance after training, is largely preserved in individuals after hemiparetic stroke [109113]. Because mi and ao do not rely on overt movement execution to activate and adaptively stimulate cortical motor representations, they represent rather promising alternative motor rehabilitation strategies for individuals that have poor residual function after stroke and that therefore cannot effectively engage with the overt practice of physically demanding motor tasks . In this case, individuals can make use of such strategies to initiate motor recovery, for instance, during the beginning of their rehabilitation, while they gradually build up their physical capacity with progressive cardiorespiratory and muscle strengthening / endurance exercises [77, 78], or simply up to a point where they are able to fully participate in overt task - specific training . Furthermore, from this point onwards, and for those who already have higher levels of residual function at the beginning of their rehabilitation, mi- and/or ao - based strategies might be used as complementary interventions to be combined with overt task - specific exercises in order to potentiate motor gains . Thus, it follows that covert practice - based alternative motor rehabilitation strategies for stroke, in comparison to overt practice - based strategies, are more compatible with the wider spectrum of functional recovery and therefore are likely to benefit a larger group of stroke survivors . Nevertheless, despite being promising, research in this arena is still in its infancy and more studies are needed before sound recommendations regarding the use of covert motor strategies such as mi and ao as motor rehabilitation strategies for stroke can be made to effectively inform clinical practice . In light of the aforementioned, researchers have started to investigate how the brain mechanisms of mi and ao interact with the effects of upper limb immobilisation . In one study, bassolino and colleagues (2013), individuals were instructed to imagine, with their eyes closed, reach - to - grasp movements being performed with their restrained arm and hand, mi group, observe the same actions through a computer screen, ao group, or watch a nature documentary with no human actions, control group . The size and excitability of the cortical motor representation corresponding to the immobilised hand were assessed with tms one day before and immediately after removal of the immobilisation . In the control group, both the size and excitability of the cortical motor representation of the restrained hand this finding is in line with the findings from the other immobilisation studies already described earlier in this paper (see section 4). In the mi group, similar changes were reported . However, in the ao group, no substantial changes were noted . Here, the excitability of the cortical motor representation of the immobilised hand was higher than in the other two groups after immobilisation, and its size remained similar to what was observed in all the three groups before immobilisation . Regarding the behavioural effects of immobilisation, it was recently suggested that they might be attenuated by mi practice before removal of the restriction . In one study, individuals had their left hand immobilised for 24 consecutive hours . After removal of the immobilisation, participants were assessed on a hand recognition task, where their goal was to identify, as quickly as possible, whether a hand displayed on a computer screen corresponded to a left or right hand . Those who did not practice mi during immobilisation showed slower response times in the task, particularly for left hand stimuli . It is not known, however, if this modulation of task performance by mi practice was mediated by changes at the level of the cortical motor representation corresponding to the immobilised hand . In addition, whether the gains in performance on the hand recognition / reaction task reported in this study extrapolate to the domain of overt motor performance also remains unknown ., that mi training was inefficient in adaptively stimulating the cortical motor representation of the immobilised hand, appears to be not only in relative dissonance with the finding from others, who have otherwise reported adaptive neural plasticity of cortical motor representations with mi training (e.g., see in section 5.1.1), but also in contradiction to jeannerod's simulation theory, which predicts adaptive stimulation of cortical motor representations with mi training . A possible explanation for this contradiction, which was indeed acknowledged by bassolino et al ., might be that the effects of mi on the motor system, particularly in terms of stimulating cortical motor representations, are influenced by the state, that is, the posture, position, and/or history of mobility, of the body parts corresponding to these representations during the imagination process . For instance, studies have shown that if the current state, in this case the posture, of a body part is congruent with the movements / actions being imagined, the size and excitability of the corresponding cortical motor representation increase to a greater extent than when the state is incongruent [117, 118]. This suggests that the afferent, proprioceptive information coming from the body part(s) involved in the imagined movements / actions may play an important role in modulating the effects of mi on the corresponding cortical motor representation(s). Therefore, according to this perspective, the immobilisation of the upper limb in the study by bassolino et al ., by maintaining the hand both in a relatively antagonistic posture and immobile for several hours, that is, in a state which was rather incongruent with the imagined reach - to - grasp movements / actions, would have compromised the effects of mi on the corresponding cortical motor representation . If this is indeed the case, it would have important implications for the use of mi in the context of motor rehabilitation after stroke, especially in cases of low - functioning upper limb hemiparesis . Here, mi training is generally used with the underlying assumption that it can adaptively stimulate cortical motor representations in the damaged cerebral hemisphere and that this might translate into improved motor function (see section 5.1.1). However, in this context, the state of the patient's paretic upper limb is sometimes largely incongruent with the movements / actions that are usually imagined during mi training, which basically consists of the mental rehearsal of functional movements of the patient's daily living, such as reaching and grasping (see for a review). Not infrequently, the paretic upper limb is found in a state that is characterised by both, a relative immobility or lack of overt voluntary movement and a spastic posture with varying degrees of sustained flexion at the fingers, wrist, and elbow . Such state, in turn, by providing the brain with reduced and/or incompatible proprioceptive information, could decrease or mask the potential of mi as a rehabilitation strategy . In keeping with this hypothesis, liepert and colleagues (2012) showed that mi of paretic hand movements increased the excitability of the corresponding cortical motor representation in stroke patients with somatosensory deficits to a much lesser degree than in patients with pure motor hemiparesis . Overall, this seemingly important influence of the current state of the body part(s) on the effects of mi on the corresponding cortical motor representation(s) could explain, at least in part, the relative inconsistency in the results from recent clinical trials of mi training after stroke (see [103, 108] for reviews). Thus, given its theoretical and clinical implications, the interaction between the effects of both mi and upper limb immobilisation on the involved cortical motor representations should be investigated in more detail . We believe the upper limb immobilisation model provides a compelling neurobehavioural framework for exploring covert motor strategies, such as mi and ao, within the context of poststroke motor rehabilitation . This is because upper limb immobilisation not only mimics the maladaptive neural plasticity patterns that are believed to contribute to the motor deficits shown by an individual after a hemiparetic stroke but, critically, also consists of a disease - free model of compromised brain function . Here, covert motor strategies can be investigated in healthy individuals for their potential to adaptively stimulate cortical motor representations, within a context of stroke - like maladaptive neural plasticity, without the influence of lesion - related confounding factors that otherwise are inevitably present in disease - based models . This opportunity, in turn, might greatly contribute to sharpening the mechanistic understanding of these strategies and thereby improve their translation to the clinical context of poststroke motor rehabilitation . Recapitulating on the role of the pattern of activity in the efferent and/or afferent neural signalling pathways targeting and/or coming from a particular body part in shaping the corresponding cortical motor representation (see section 2.1), a recent study tested in healthy individuals the interaction between peripheral somatosensory stimulation (pss) and upper limb immobilisation . Complementing their previous findings on the effects of immobilisation on both the excitability of homologous cortical motor representations and the interhemispheric interactions between them, avanzino et al . (2014) showed that a form of proprioceptive stimulation can largely attenuate the maladaptive neural plasticity that is induced by upper limb immobilisation . Specifically, they found that intermittent vibration of a muscle from the immobilised hand delivered throughout the period of immobilisation was able to prevent large decreases in the excitability of the corresponding cortical motor representation and to abolish both, increases in the excitability of the homologous representation in the opposite hemisphere and changes in the interhemispheric balance between the two homologous cortical motor representations . The findings from this latter study, when taken together with those from previously described studies investigating covert motor strategies during immobilisation, invoke the idea that different interventions might be delivered in combination during upper limb immobilisation in order to maximize prevention of maladaptive / promotion of adaptive neural plasticity in the brain's motor system, much like with the use of adjunctive therapies in association with task - specific exercises during poststroke motor rehabilitation (see section 3). Here, a potential manipulation could be, for instance, to combine covert motor strategies with brain stimulation (bs) and/or pss techniques during upper limb immobilisation (figure 2(b)). It is likely that the effects of such strategies, in terms of adaptively stimulating cortical motor representations, would be strengthened by concomitant bs, delivered to either one or both motor cortices, and/or pss of the immobilised body part(s). It was shown that observation of right hand movements combined with sensorimotor electrical stimulation of the median nerve at the level of the right wrist increased the excitability of the cortical motor representation of the involved hand, an effect that was not present when the two interventions were delivered separately . It remains to be tested, however, whether a similar manipulation would result in a potentiation of the effect of ao during upper limb immobilisation . Advances in our understanding of the neural plasticity that occurs after hemiparetic stroke, both adaptive and maladaptive, have contributed to the formulation of theories of motor recovery after stroke . Such theories identify the processes that are likely to promote motor recovery, for instance, of poststroke upper limb hemiparesis, and that therefore might be targeted by neurorehabilitation efforts . Contemporary motor rehabilitation strategies target these processes, but because they are essentially centered in the overt practice of physically demanding task - specific exercises, they do not easily translate into benefit for stroke survivors sustaining low - functioning upper limb hemiparesis . For these individuals, motor rehabilitation options are currently limited . Mi and ao are two examples of covert or cognitive motor strategies that have a great potential to translate into alternative motor rehabilitation strategies for stroke . Because they can adaptively stimulate cortical motor representations in the absence of overt movement, mi- and ao - based training strategies have a greater potential for benefiting the wider spectrum of functional recovery after stroke, including those individuals with poor residual function . In this paper, we provided theoretical and empirical evidence that an upper limb immobilisation - based neural plasticity model, by capitalising on current theories of motor recovery after stroke, the shortage of overt movements, and a disease - free condition of compromised brain function, provides a very attractive neurobehavioural template for exploring these strategies within this context . The findings from the immobilisation studies reviewed here, particularly those concerning the effects of immobilisation on the interhemispheric interactions between homologous cortical motor representations, have important implications for a particular contemporary motor rehabilitation strategy addressing poststroke upper limb hemiparesis . Cimt falls within one of those two categories of interventions that were presented in this paper (see section 3). These are (1) repetitive and progressive task - specific training with the paretic upper limb for several hours a day, usually for a period of 2 weeks; (2) a transfer package made of behavioural techniques used to promote transfer of the gains obtained during the treatment period in a research and/or clinical setting to the individual's real - world environment; and (3) constraining use of the paretic upper limb through immobilisation of the contralateral, less - affected limb . This particular therapy has proven to be an effective intervention for improving paretic upper limb function after stroke and is currently recommended as the treatment of choice for those individuals with relatively high levels of residual function . Despite the fact that cimt has been extensively investigated during the past few years, the mechanisms mediating treatment success with this intervention are still relatively poorly understood (see for a discussion). One influential proposal has been that functional improvements after cimt are consequential to the massed overt motor practice with the paretic upper limb, which, in turn, contributes to both reversal of the learned nonuse phenomenon and adaptive neural plasticity, that is, increases in the size and/or excitability, of the corresponding cortical motor representations . It follows from this proposal that the relevance of immobilising the less - affected upper limb for promoting motor gains with the more - affected, paretic limb is often underestimated . More commonly, an indirect, relatively trivial role is attributed to the immobilisation element of cimt, for example, that it serves only as a constraining instrument employed simply to encourage use of the paretic upper limb . As taub and colleagues (1999) commented on the relevance of the immobilisation element of cimt, there is thus nothing talismanic about use of a sling or other constraining device on the less - affected limb . Any technique that induces a patient to use an affected limb many hours a day for a period of consecutive days should be therapeutically efficacious . This factor is likely to produce the use - dependent cortical reorganization found to result from ci therapy (23, 58, 59) and is presumed to be the basis for the long - term increase in the amount of use of the more - affected limb . As already pointed out by others (e.g., see), recent findings from upper limb immobilisation studies suggest, however, that a more sensible appreciation of the contribution of the immobilisation element of cimt would be that such manipulation might otherwise have a more direct, meaningful role in mediating treatment success, at least in theory . For example, it is possible that, at least in some individuals, the effects of immobilisation of the less - affected upper limb during cimt would be superimposed to those effects induced by the intensive motor training with the paretic upper limb . More specifically, as long as a condition of true immobilisation, that is, of substantially reduced usage, of the less - affected upper limb is present, it would decrease activity of the contralesional motor cortex, which, in turn, could contribute to reducing transcallosal inhibition from this region towards the homologous area in the affected hemisphere . As a consequence, this could attenuate interhemispheric imbalance and facilitate ipsilesional activation and hence boost massed practice - induced adaptive neural plasticity (see rebalancing theory in section 2.2). Since its original conceptualisation, cimt has taken many different forms, each of which containing different combinations and/or durations of the three elements of the intervention, which are task - specific training, transfer package, and immobilisation of the contralateral, less - affected upper limb [127129] (see for a review). When considering whether or not to include the immobilisation element of cimt in the rehabilitation program, the above - mentioned consideration should be taken into account, but also the pros and cons of restraining movements of the patient's less - affected upper limb should be carefully weighted . On a different note, with the advent of noninvasive brain stimulation (bs) techniques during the past years, it has become possible to develop neural plasticity protocols that are able to induce relatively predictable changes in the excitability of selected brain regions and that therefore might be used to improve the individual's behavioural capacity under certain conditions . The motor cortex, in particular, has been a major target of such protocols . Here, bs has been used to either directly induce adaptive neural plasticity of cortical motor representations in healthy individuals with excitatory stimulation and thereby improve their motor learning or adaptively modulate neural plasticity patterns after hemiparetic stroke with excitatory and/or inhibitory stimulation and thereby improve the individual's motor recovery (see section 3 and also [66, 130] for reviews). More recently, neural plasticity research has shown that it is possible to modulate the response of the motor cortex to neural plasticity protocols, to both those exogenously induced through bs and endogenously induced protocols, such as motor learning, by manipulating its activity before delivery of such protocols . In other words, the strength of an excitatory or inhibitory neural plasticity protocol that targets the motor cortex might be either increased or decreased depending on the history of activity of this area . Basically, if activity in a brain region or neuronal network is high, then the strength of a subsequent excitatory protocol is decreased, while the opposite is true for a subsequent inhibitory protocol . Conversely, if activity in that brain region or neuronal network is low, then a subsequent excitatory protocol is enhanced, while the opposite is true for a subsequent inhibitory protocol . An endogenously induced excitatory neural plasticity protocol that targets the motor cortex can be potentiated by decreasing activity / excitability of the targeted motor cortex with a long - term depression- (ltd-) inducing paired associative stimulation (pas) protocol delivered before the learning paradigm . Motor learning is thought to improve the individual's motor capacity by, among other processes, inducing long - term potentiation (ltp) and thereby adaptive neural plasticity in their motor cortex (see section 2.1). In the study by jung and ziemann, it is likely that the ltd - inducing pas protocol delivered before motor practice increased, via metaplasticity mechanisms, the likelihood for subsequent ltp in the involved motor cortex and cortical motor representation(s), hence potentiating motor learning . Recently, this finding has prompted others to extrapolate the concept of metaplasticity to the context of motor rehabilitation after stroke . Interestingly, in a proof - of - principle study, challenging the standard approach in bs - based poststroke motor rehabilitation (e.g., see section 3 and figure 1(b)), di lazzaro and colleagues (2013) found that, in general, inhibitory, activity - decreasing bs of the ipsilesional motor cortex followed by physiotherapy exercises improved the motor outcomes of patients more than sham bs . The results from these two latter studies, when taken together with the findings reported in this paper concerning the effects of upper limb immobilisation on the motor cortex, might spark rather provocative speculations . As demonstrated by rosenkranz et al . (2014) (see end of section 4), apart from decreasing the excitability of the corresponding cortical motor representations in the contralateral motor cortex, upper limb immobilisation also seems to alter neural plasticity mechanisms within these representations, such that the larger the decrease in their excitability induced by the restriction, the greater their sensitivity to subsequent ltp - like processes . This suggests that, like bs, but with the advantages of being much cheaper and technically simpler, upper limb immobilisation could also be used as a noninvasive neural plasticity protocol to decrease motor cortex activity / excitability and, within a context of metaplasticity, improve subsequent motor training . For instance, it can be speculated that, in healthy individuals, immobilisation of the upper limb for a certain period of time which is sufficient to substantially decrease the excitability of the corresponding cortical motor representations in the contralateral motor cortex could perhaps facilitate / enhance subsequent motor learning with that limb by increasing the likelihood for ltp in the involved representations . In the same vein, now within the context of poststroke motor rehabilitation, it can also be speculated that a period of immobilisation of the patient's paretic upper limb before a physiotherapy session could boost subsequent practice - induced adaptive neural plasticity of the corresponding cortical motor representations . This, in turn, could eventually enhance rehabilitation outcomes . Despite their rather provocative and speculative nature, the findings discussed previously suggest that such predictions are, at least theoretically, valid . Before upper limb immobilisation can be consolidated as a neural plasticity model / protocol in humans, some issues remain to be addressed by future research . First, most of the studies, if not all, on upper limb immobilisation have been conducted with young participants . It would be important to also perform these investigations with aged populations, especially within the context of translational neurorehabilitation research, as the vast majority of stroke survivors are elderly . Second, there is no consensus yet on a possibly ideal immobilisation time to induce the reported changes in the motor system, both those that are measured at the level of the motor cortex and those expressed behaviourally . For instance, studies have used restriction periods ranging from hours, for example, 6, 8, 10, 12, and 24 hours, to days in humans and to months in animals . Determining the optimal length of immobilisation is critically important to minimise not only costs, but also potential ethical issues that might be present, particularly in long - term immobilisation studies . In this case, the question of whether the effects of immobilisation on the brain's motor system can be potentiated, and perhaps accelerated with exogenous manipulations, such as noninvasive bs, should also be given consideration.
The golgi complex serves as the central station in the biosynthetic pathway, where proteins are sorted for their different destinations, such as various domains of the cell surface and the endosomal lysosomal system . This delivery of cargo proteins from the golgi complex to their target compartments is carried out by dynamic, membrane - bound organelles that are frequently called either transport carriers or post - golgi carriers (pgcs) (luini et al . As these pgcs have an important role in the process of intracellular transport, their morphology, living dynamics and molecular compositions have become the focus of significant interest, particularly over the last few years . These pgc transport organelles were originally defined through the development of green fluorescent protein (gfp) technology and living - cell imaging (lippincott - schwartz et al . 2000), whereby the first fluorescent cargo protein that was followed in living cells revealed a new world of images of these relatively large and highly dynamic structures that travel from the golgi complex to the plasma membrane (wacker et al . 1997 1998). Then, with time, the list of cargo molecules that could be visualized in vivo expanded, further exposing the unexpected complexity of the post - golgi transport pathways . Finally, further technical advances resulted in the combination of video and electron microscopy (correlative light - electron microscopy, clem), which provided the means to define the morphogenesis of these pgcs at the ultrastructural level . As a result of these studies we now know that pgcs arise from specific membrane domains of the golgi complex that lack resident golgi enzymes, forming what are known as the pgc precursors (hirschberg et al . The shapes and sizes of pgcs that even carry the same cargo can vary across a wide range, and under the light microscope, most of them are seen to be clearly larger that plasma - membrane associated clathrin vesicles and 100-nm fluorescent beads (hirschberg et al . 1998). The smaller pgcs usually have a size of 300400 nm, although some of the larger ones can reach several microns in length . Video microscopy has also revealed that many of these carriers appear globular, although they frequently stretch out into tubular shapes during their translocation through the cytosol; thus pgcs have frequently been termed as pleiomorphic structures . In transporting their cargoes to the correct acceptor compartments, however, carriers can also form and support post - golgi transport without microtubules, although the correct targeting of their cargo proteins is usually compromised under these conditions (rindler et al . Finally, the life cycle of a pgc can be schematically imagined to consist of three stages: (1) its formation (which can in turn be further divided into several substages; see below); (2) its transition through the cytosol; and (3) its docking and fusion with the target membrane (polishchuk et al . The first of these steps, the formation of the pgc, appears to be the most complex, and it also probably remains the least understood . Since the question of the morphogenesis of a pgc is closely intertwined with that of the structure of the organelle from which it originates, the trans - golgi network (tgn), both of these issues will now be discussed in an integrated fashion . The process of pgc morphogenesis has been characterized in detail using video and electron microscopy, as well as with the combination of these techniques (clem). As indicated above, this process comprises the formation, the extrusion and the fission of the export domain from the tgn, thus generating the free carrier (polishchuk et al . The first step (the formation of the tubular export domains) also includes the segregation of the cargo proteins from the golgi resident enzymes . These events appear to be common to the proteins directed towards different post - golgi compartments, such as basolateral plasma membrane (hirschberg et al . 1998; white et al . 2001; polishchuk et al . 2003), the apical plasma membrane (keller et al . 2001) and the endosomal lysosomal system (puertollano et al . These export domains usually contain tgn markers; i.e. They are part of the tgn (polishchuk et al . 2003; puertollano et al . 2003). Of note, these three post - golgi compartments are the ones that have been best characterized for the pgcs leaving the tgn, with the total number and types of pathways for cargo exit from the tgn at present not known, although there are at least six, and probably more (rodriguez - boulan et al . 2005). Fig . Subsequent frames extracted from a time - lapse sequence illustrating the different stages of pgc biogenesis (arrows): a formation of the tubular domain containing the cargo vsvg - yfp and devoid of the golgi - resident protein galatosyltransferase - cfp . Arrows indicate tubular pgc precursor that has been pulled out of the tgn area of the golgi complex formation of post - golgi transport carriers . Subsequent frames extracted from a time - lapse sequence illustrating the different stages of pgc biogenesis (arrows): a formation of the tubular domain containing the cargo vsvg - yfp and devoid of the golgi - resident protein galatosyltransferase - cfp . Arrows indicate tubular pgc precursor that has been pulled out of the tgn area of the golgi complex although the existence of the tgn has been known for many years (griffiths and simons 1986), its precise structure and identity still remain to be ascertained . In one view, which predominated in the field until recently, the tgn was considered to consist of essentially an anastomosing tubular network that emanates from (or that results from the breakdown of) the trans - most golgi cisterna, and that projects mainly in the trans direction (griffiths et al . Rambourg and colleagues have thoroughly described the tgn in several different cell types . In these descriptions, this trans - most cisterna also tends to peel off from the rest of the golgi stack, and together with the other morphological characteristics of the golgi stack, this is a feature that is suggestive (clermont et al . 1995) of the cisternal progression - maturation trafficking model (bannykh and balch 1997; mironov et al . 1997; bonfanti et al . 1998; losev et al . 2006; matsuura - tokita et al . Thus, in a simple version of this scheme, the tgn would result from the final stages of maturation of the golgi cisternae . This would include the partial transformation of this trans - most cisternae into a tubular network, and the various carriers that leave the tgn for their different destinations would all originate form different domains of this tubular network . A more complex and more recent view of the organization at the trans face of the golgi complex comes from studies using electron microscopy tomography of cryofixed, freeze - substituted cells (ladinsky et al . 1999, 2002). These studies have indicated that the tgn derives not only from the last trans cisterna, but instead from the three trans - most cisternae (roth et al . 1985), from where tubules emanate into the trans space of the golgi stacks . Remarkably, only the trans - most cisterna exhibits clathrin - coated buds, with the other buds appearing to have different, yet - to - be identified, types of coats (ladinsky et al . 1999). Thus, only the trans - most cisterna would be responsible for the clathrin- and ap-1-dependent trafficking towards the endo - lysosomes, and although all of these three trans - most cisternae of the golgi stacks would serve as the classical tgn, each of them could be specialized in the packaging and export of specific cargo proteins . This might have important functional and mechanistic implications for the proteins sorting and export process . Another remarkable tgn feature that has been revealed by these tomographic studies is that these three trans - most cisternae can intercalate with the cisternae of the endoplasmic reticulum (er) (ladinsky et al . 1999, 2002). These have been proposed to have important roles in the transfer of lipids (through specialized contact sites) between the er itself and the golgi complex . It will be important to establish whether the morphological discrepancies described above reflect fundamental differences in the organization of the export of cargo in different cell systems . Indeed, it is possible that the first model (the tgn as a network resulting from the tubular disassembly of the last trans - most cisterna) might actually only represent a special case of the more complete three - trans - cisterna model, and that both of these two different models of tgn may apply, depending on the cell type and the functional conditions . In support of this possibility, it is clear that the tgn can vary significantly in both size and composition across different cell types . For example, cells with a well - developed endo - lysosomal system and without secretory granules exhibit an extensive tubular tgn, while the tubular component of the tgn is reduced in cells that are specialized in regulated secretion (clermont et al . 1995). This appears to occur in the latter because most of the tgn membranes are used for the packaging of secretory granules, and the tubules only form the thin bridges between the granule precursors . Such an organization simplifies the release of the granules, which occurs through the rupture of these tubular elements (clermont et al . Similarly, human fibroblasts have large collagen - containing distensions that are connected to tubular elements in the trans - golgi area (polishchuk et al . Thus, the morphology and size of the tgn apparently depend on the predominant type and amount of cargo protein departing from the golgi complex . The morphology of the tgn depends also on its secretory status . When exit from the tgn is blocked by a lowering of the temperature to 20c in the presence of abundant cargo (matlin and simons 1983), the volume and surface areas of the tgn increase greatly (griffiths et al . 1989); while in the absence of cargo, the 20c block results in all three of the trans - most cisternae producing bulging exit domains (ladinsky et al . 2002). At physiological temperatures, three - dimensional (3d) analyses of the golgi stacks have revealed well developed, tubular reticular, tgn - like membranes at the trans side of the golgi complex in actively secreting cells (trucco et al . 2004), while the tgn is nearly absent in quiescent cells (trucco et al . Thus, the tgn is a very dynamic structure, the shape and size of which is potently regulated by the extent of traffic flowing through it . To discover the regulatory mechanisms is an important challenge for the future . Another challenge will be to define whether the three - cisterna organization really underlies a functional specialization of these cisternae in the sorting and packaging of different cargo classes, as suggested by the presence of clathrin buds only on the last cisterna of the golgi stack . So far, the lack of immunolabeling studies does not allow determining whether different trans cisternae really do contain different transport proteins . In addition to being an organelle of the biosynthetic pathways, the tgn is involved in endocytic transport routes (griffiths and simons 1986; mellman and simons 1992; pavelka et al . 1998). Several studies with different cell types showed that the tgn and golgi stacks can contain plasma - membrane constituents and internalized materials (stoorvogel et al . Could well introduce a further level of structural complications, since the recycling endosomes, in particular, reside in the golgi area and are tubular in nature . In conclusion, both the dynamics and the structure of the tgn are variable and incompletely defined, thereby presenting an additional layer of difficulty to our understanding of the biogenesis of the carriers that depart from this organelle . There are two key aspects to the formation of the pgc precursors: their morphogenesis at the tgn; and the segregation between different types of cargo proteins that are targeted for different destinations, as well as between the golgi - resident proteins, such as the golgi enzymes . Regarding pgcs morphogenesis, in principle, the tubular carrier precursors might form through the mechanical pulling force exerted by microtubule - based motors on a flatter, parent, membrane domain (e.g., a golgi cisterna). This has been shown to be mechanistically possible for both artificial and natural membranes (roux et al . If this is the case, the pgc precursors would be expected to be essentially simple linear tubular structures (roux et al . The second possibility is that these precursors are actually formed from the tubular subdomains of the tgn, which are generated prior to docking and extrusion by microtubules . In this case recent studies have provided compelling support in favor of the latter model (polishchuk et al . First, both free pgcs and their precursors at the tgn comprise complex tubulo - reticular structures, which have often been described as the main component of the tgn in many cell types (clermont et al . For example, pgcs carrying vsvg show a mostly tubular morphology, and can have a complex structure that even contains clearly visible fenestrae (polishchuk et al . 2003); this would be expected of membranes that derive from protrusions of the tgn . Indeed, pgc precursors that have been visualized using clem, appear to be comprised of tubular segments that are interconnected with complex branching and fenestrated membranes; they are also seen to be continuous with the parent membranes of the golgi stack (polishchuk et al . Similarly, carriers containing the apical cargo protein hemagglutinin (ha) frequently have a tubular morphology as well as ha - positive domains at the tgn (puertollano et al . Thus structural similarities between pgc precursors and the tgn appear to be a common feature of different types of pgcs . This strongly suggests that pgcs form via the fission of an entire precursor domain (or a large part thereof) from the rest of the tgn membranes . Thus the question regarding the mechanism by which the originally flat golgi membranes are converted into highly bent, tubular essentially, this can be achieved by one of two mechanisms: either by the action of proteins that can bend these membranes into tubules, in a manner that is possibly similar to that of amphiphysin, endophilin, sorting nexins and others (antonny 2006), or via the alteration of the lipid composition of these trans - golgi membranes, which can be mediated, in turn, by several processes . For instance, changes in lipid content, and hence membrane curvature, can be modulated via the lipid - metabolizing enzymes that reside at the golgi complex (for review, see de matteis and godi 2004; luini et al . . The transmembrane or inter - organelle transfer of lipids can contribute to the generation of particular lipid environments in the membranes of the tgn . In this respect, it is important to note that numerous contact sites between the er and the trans - cisternae of the golgi complex have been detected by electron microscopy tomography (ladinsky et al . Such contact sites can be explored for lipid transfer between the er and trans - golgi that is mediated by specific lipid - transfer proteins . Also, since the cisterna - like morphology of golgi compartments can be stabilized by large polymers of golgi enzymes (nilsson et al . 1996), the loss of oligomerized golgi enzymes in the trans - golgi should, in principle, favor cisterna - to - tubule transformation of cargo - containing membranes . Moreover, this process can be accompanied by the loss of the stacking mechanisms at the trans side of the golgi complex . Here, grasp65 and grasp55 are two proteins that have been suggested to be involved in the maintenance of cisterna juxtapositioning, and they are located mainly at the cis and medial golgi, rather than the trans - golgi (barr et al . So this intercisternal glue may be gradually lost as a cisterna progresses towards the trans pole of the golgi complex . This has been confirmed both in mammals and yeast by the observation that the trans - most cisterna frequently peels off from the main golgi stack (clermont et al . It is possible that more than two or more mechanisms act in synergy to provide this transition from flat golgi cisternae to the tubulo - reticular tgn morphology . In addition to the formation of tubular domains at the exit face of the golgi complex, this is also the level at which cargo proteins that are directed to different post - golgi destinations should be sorted . The classical view in the membrane transport field implies that sorting at the tgn (as well as throughout the whole secretory pathway) is driven mainly by the coat - adaptor - protein machinery, which interacts specifically with amino - acid signals of certain transmembrane cargo proteins; this then provides the mechanical force for budding and fission of transport vesicle (mellman and warren 2000). This holds true for the endo - lysosome - directed carriers that have been thoroughly characterized . These carriers consist of clusters of clathrin - coated buds that are connected by tubular regions, and thus exhibit a grape - like structure . In contrast, pgcs carrying a cargo like the g - protein of vesicular stomatitis virus (vsvg) form in a coat and an ap - independent manner . Both pgcs and their precursors do not show -cop or -, - and -adaptins at their membranes (polishchuk et al . 2003). Other adaptors, such as the ggas, are excluded from vsvg carriers as well (puertollano et al . Similarly, coats and adaptors have also never been detected on pgcs that are carrying proteins to the apical surface in polarized cells (kreitzer et al . Thus, these carriers should form either by virtue of some still - unknown adaptors that cannot yet be visualized by electron microscopy, or by their association with specific lipid microdomains that are involved in sorting (schuck and simons 2004). This might be the case for proteins directed to the apical surface in polarized epithelial cells, the concentration of which at the tgn appear to be through their partition into cholesterol- and sphingolipid - rich membrane domains that are known as rafts (schuck and simons 2004). Observations in living cells have revealed that the fission of pgcs frequently coincides with mechanical pulling of carrier precursor from the tgn along microtubules . Apparently, the pulling force which the molecular motors such as kinesin (see below) can apply to the tgn membranes is important to facilitate the extension of pgc precursors from the golgi body and for the later fission of the pgc (kreitzer et al . 2003). In cell - free systems, the addition of kinesin to golgi membranes (and even to liposomes) together with microtubules induces the formation of tubule - like membranes that are similar to pgc precursors (roux et al . 2002), while a block of kinesin function by microinjection of an inhibitory antibody (kreitzer et al . 2000) or expression of the headless kinesin mutant (nakata and hirokawa 2003) prevent pgc formation from the golgi complex . Kinesin has been seen to be associated with the tip of pgc precursors, although it can also attach to other points along the pgc precursor membrane (polishchuk et al . The movement of kinesins along microtubules can then create tension within the pgc precursor that will facilitate the fission process . Indeed, based on in vitro data, membranes under tension have recently been proposed to have an important role in fission (roux et al . However, pgcs can also form when microtubules have been destroyed by nocodozole treatment; in this case, the pulling force to create membrane tension in fission - prone regions might be provided by actin motors (warner et al . Live cell imaging and clem have also shown that fission does not take place randomly along the membranes of pgc precursors . In this case, which regions of a pgc precursor can be defined as prone to fission? Our data suggest that fission usually takes place at the thinnest parts of the pgc precursor (fig . 2a), which at the electron microscopy level corresponding to thin tubular segments of membranes (polishchuk et al . In contrast, fission does not take place at the tgn regions with a complex morphology (i.e., in those containing tubular networks and branching tubules, or in thick vacuolar regions). Obviously, the precise points of fission will define not only the compositions of the pgc carriers, but also their morphology . If fission occurs close to the tip of the tgn tubule, a carrier will be smaller in size . In contrast, larger pgcs can form by cleavage at the base of the pgc precursors (fig . Similarly, endosome - directed pgcs can apparently detach from the tgn as simple cargo - containing vesicles if the fission occurs at the neck of the clathrin - coated buds (fig . However, many clathrin - positive pgcs have a grape - like morphology (tubule with several buds), suggesting that entire chunks of tgn membranes containing 23 clathrin - coated buds can be cleaved from the golgi complex (polishchuk et al . The fission (red line) of the carriers occurs at the thinnest parts of the pgc precursor, which correspond to thin tubular segments of the tgn membrane at the electron microscopy level . In contrast, fission does not take place at the tgn regions with a complex morphology (i.e., containing tubular networks and fenestrae, or in thick vacuolar regions). If fission occurs close to the tip of a pgc precursor, the carrier will be smaller in size (1). In contrast, larger pgcs can be formed by cleavage at the bottom of a pgc precursor (2). B pgcs directed to endosomes detach from the tgn as simple clathrin - coated vesicles if fission (red line) occur at the neck of the clathrin - coated bud (1). Alternatively, entire chunks of the tgn membrane containing 2 - 3 clathrin - coated buds can be cleaved from the golgi complex fission of post - golgi transport carriers . The fission (red line) of the carriers occurs at the thinnest parts of the pgc precursor, which correspond to thin tubular segments of the tgn membrane at the electron microscopy level . In contrast, fission does not take place at the tgn regions with a complex morphology (i.e., containing tubular networks and fenestrae, or in thick vacuolar regions). If fission occurs close to the tip of a pgc precursor, the carrier will be smaller in size (1). In contrast, larger pgcs can be formed by cleavage at the bottom of a pgc precursor (2). B pgcs directed to endosomes detach from the tgn as simple clathrin - coated vesicles if fission (red line) occur at the neck of the clathrin - coated bud (1). Alternatively, entire chunks of the tgn membrane containing 2 - 3 clathrin - coated buds can be cleaved from the golgi complex another factor that might be important for pgc shaping is the type of cargo that is embedded in the pgc . For example, procollagen - i usually forms quite large aggregates that are visible within 300400-nm - diameter membrane distensions of the golgi membranes . As a consequence, similar distensions have been detected in most collagen - containing pgcs (polishchuk et al . 2003; canty et al . Different members of the kinesin superfamily (kamal et al . 2000; nakata and hirokawa 2003; teng et al . 1999), have been shown to drive post - golgi transport of specific cargo to various destinations . This high fidelity of cargo selection by molecular motors at the tgn and their further delivery to the correct surface or intracellular domain may be regulated by interactions of motor protein directly with the cargo (kamal et al . 2000; teng et al . 2005) or with components of the sorting machinery at the tgn (nakagawa et al . 2000). As an example, transport of ha and annexin 13b to the apical surface in epithelial cells relies on raft - associated motor kifc3 (noda et al . Kif13a operates in the other post - golgi route that is used for the transport of the mannose-6-phosphate receptor (nakagawa et al . 2000). A number of neuronal proteins, including bapp, gap43 and vamp-2, require kif5 for their correct targeting (nakata and hirokawa 2003), while the microtubule minus - end - directed motor dynein has been shown to support rhodopsin transport in rod photoreceptors (tai et al . The simplest would use a direct interaction between the motor and a specific domain of a cargo protein, as is seen for the dynein light chain and the cytoplasmic tail of rhodopsin (tai et al . Alternatively, adaptor proteins could serve as a bridge between a motor and its cargo . For example, kif13a transports the mannose-6-phosphate receptor through its interaction with the ap-1 complex (nakagawa et al . 2000). Finally, both motor and cargo could associate with the same specific lipid microdomain, as for instance, for kifc3 and the apically targeted annexin xiiib . Another issue that needs to be addressed is whether any of these sorting processes take place in the pgcs while they are moving toward their acceptor compartment . This happens, for example, with the maturation of secretory granules when the mannose-6-phosphate receptor is concentrated and sorted from the secretory granules by clathrin - coated vesicles (klumperman et al . So several strategies have been used to determine whether similar sorting events happen with pgcs . Mature exocytic carriers can be arrested before their fusion with the plasma membrane either by microinjection of an anti - nsf antibody or by treatment with tannic acid (which fixes the plasma membrane but does not penetrate inside the cell). In contrast to secretory granules, the comparison of mature and newly formed pgcs did not reveal significant changes in either their ultrastructure or their composition (polishchuk et al . Similarly, mature golgi - to - endosome carriers were accumulated in cells upon endosome inactivation . However, they did not show any significant transformation, except for a very moderate reduction in the area covered by clathrin (polishchuk et al . Live - cell imaging of subconfluent mdck cells has shown that pcgs that initially contained both a basolateral marker (vsvg - cfp) and an apical marker (gpi - yfp) did not sort out either of these cargoes into any separate structures, and instead delivered both of the proteins to the plasma membrane (polishchuk et al . Gpi - gfp was then sorted from the basolateral surface to the apical membrane through transcytosis (polishchuk et al . The partitioning of two proteins from a common pgc into separate carriers has also been reported (jacob and naim 2001). This suggests that sorting from the pgc may exist, but that it should depend on the nature of the cargo proteins being transported . The complexity of sorting events in the post - golgi space appears to be even worse since the discovery that certain cargoes may pass through the endosomal compartments before their arrival at the plasma membrane . Sporadic reports in the past have suggested that some secretory proteins do not move from the golgi complex directly to the plasma membrane, but instead pass through an endocytic intermediate on their way to the cell surface (leitinger et al . This indirect through - endosome delivery of cargo to the plasma membrane might be also facilitated by the intimate association of tgn membranes with number of endocytic compartments in the perinuclear area of the cell (marsh et al . The list of the proteins using this pathway has been recently updated, and it has now been shown that in mdck epithelial cells, vsvg, the ldl receptor and e - cadherin can be detected in the endosomes before their exit to the plasma membrane (ang et al . 2004; lock and stow 2005). The first is whether this transport route is ubiquitous (i.e., does it exist in different cells?). The second question is whether different cargoes move through the same endosomal compartment on their way to the cell surface in epithelial cells? A number of proteins (such as vsvg and the ldl receptor) have been reported to use a rab8-positive sub - population of endosomes as an intermediate station on their way to the basolateral membrane in epithelial cells (ang et al . These proteins require the ap-1b adaptor complex to be properly sorted from these rab8 endosomes towards the basolateral surface domain (ang et al . 2003). Other cargoes (such as e - cadherin, for example) move to the plasma membrane through a rab11 endocytic compartment (lock and stow 2005). It remains to be determined, however, whether there is any cross - talk between these rab8- and rab11-dependent routes . This possibility apparently exists, since rab8 and rab11 endosomes are both accessible to transferrin (ang et al . Finally it is important to clarify as to what extent this through - endosome transport route is used by different cargo proteins in epithelial cells . This issue has been partially addressed by the silencing of the 1b subunit of the ap-1b adaptor complex, which resides at the endosomes and is required for the correct delivery of many basolateral proteins . The use of rna interference has revealed, however, that a number of cargoes (such as transferrin and fc receptors, for example) can be targeted correctly even in the absence of ap-1b, which suggests that these proteins can move directly from the golgi complex to the basolateral surface without crossing the endocytic routes (gravotta et al . The existence of more than one basolateral pathway has also been supported by the observation that transport of different basolateral proteins can be selectively regulated by different pkd isoforms (yeaman et al . Thus, further efforts need to be made to understand to what extent an endocytic post - golgi compartment is involved in the sorting and transport events of cell - surface proteins . The extensive characterization of pgc morphology by video and electron microscopy has provided a framework for the positioning of the molecular machineries in the morpho - functional maps of various trafficking segments . At the same time, many molecular players in tgn - to - plasma membrane transport have been identified . The attribution of each of the molecular components to each of the pathways is probably now the main challenge . We believe that the development of specific assays will significantly assist in the achieving of this objective . Video microscopy of gfp - tagged cargo proteins has allowed us to evaluate the process of pgc formation, as well as the speed and directionality of pgc movement through the cytosol . 2006) or horse - radish - peroxidase - based endosome immobilization (ang et al . These conditions allow us to trap mature pgcs and to compare their compositions to newly formed carriers and the tgn membranes . Moreover, preferential sites of pgc docking and fusion can also be easily identified in this way . The combination of these experimental approaches with specific molecular inhibitors now allows us to attribute a protein of interest to one of the steps in post - golgi transport.
Although advances in percutaneous coronary intervention (pci) have dramatically improved the outcomes of ischemic heart disease, procedure - related complications such as device fractures or dislodgements are also increased . Guidewire fractures are very rare with an incidence of approximately from 0.1% to 0.2% of cases.1)2) although remnant guidewires can lead to life - threatening complications,3)4) and recently, percutaneous retrieval techniques are considered good therapeutic options, there have been several reports on the remnants being left in coronary artery and aorta without any serious adverse outcomes.5)6) we describe the case of fatal subacute stent thrombosis caused by guidewire fractures with retained remnant eleven days after the index procedure . A 72-year - old woman was referred from another hospital for coronary intervention of distal left main disease . The electrocardiogram (ecg) showed normal sinus rhythm without any changes in abnormal st segment . Her left coronary angiogram (cag) showed tight luminal narrowing at the distal left main coronary artery (lm) with preserved distal coronary flows {thrombolysis in myocardial infarction (timi) grade 3} (fig . The left coronary artery was engaged with the extra back up (ebu) 3.5 guiding catheter (6 fr, medtronic, minneapolis, mn, usa) with side hole via right radial artery . Two 0.014 inch guidewires were positioned into the left anterior descending artery (lad) (balanced middle weight, abbott vascular, santa clara, ca, usa) and the left circumflex artery (lcx) (runthrough, terumo, tokyo, japan). Predilatation and balloon kissing were done with 2.520 mm balloon (trek, abbott vascular, santa clara, ca, usa) in lad up to 12 atm and 2.020 mm balloon (genoss, suwon, korea) in lcx up to 10 atm . We implanted a 2.7518 mm everolimus - eluting stent (xience prime, abbott vascular, santa clara, ca, usa) at the proximal segment of lcx with minimal protrusion into the lm, and which was crushed using 2.520 mm trek balloon . We also deployed a 3.018 mm everolimus - eluting stent (xience prime, abbott vascular, santa clara, ca, usa) from the lm trunk to the proximal segment of lad across the ostium of lcx . We were about to insert intravascular ultrasound (ivus) catheter into the lcx after successful re - wiring into the lcx with runthrough guidewire, and then, we realized that the guidewire was kinked at small branch of lcx (fig . Even though the wire was broken at the junction of hard shaft and soft wire tip, we could see the elongated and uncoiled wire remnant swinging in the ascending aorta on cine view (fig . We changed guiding catheter for a bigger one (7 fr, ebu) via right femoral approach . Several attempts for removal of remnant wire by using amplanz gooseneck snare (6 fr, 15 mm, loop snare catheter, ev3, plymounth, mn, usa) was not successful (fig . We tried again by using endoscopic biopsy forceps (6 fr, 1550 mm, olympus, tokyo, japan) (fig . Eventually, we caught the broken wire and could remove a part of the wire (fig . 3), leaving the soft tip in the small branch (fig . 4a and b). The radiopaque remnant was fixed to the vessel wall by an additional stent implantation (2.7523 mm, xience prime, abbott vascular, santa clara, ca, usa). Elongated and un - coiled filaments were not seen within lm and proximal lcx on cine view . However, the uncoiled filaments were identified within the distal lm and proximal lcx on ivus (fig . After seven days, the patient was discharged with triple antiplatelet agents including aspirin, clopidogrel, and cilostazol . Four days later, the patient revisited the emergency department with acute chest pain in shock state . Ecg showed st - segment elevation in lead avr and avl and st - segment depression in lead ii, iii, avf, and v 4 - 6 . Shortly afterwards percutaneous cardiopulmonary support (pcps) was applied at the bed side right away, and then, the patient was sent to the coronary catheterization laboratory . Bed side echocardiography demonstrated severe lv systolic dysfunction with about 20% of the ejection fraction . Emergency cag was performed immediately and it revealed that lm was totally occluded due to subacute stent thrombosis (fig . 5). The timi flow was recovered to grade ii after aspiration thrombectomy and balloon angioplasty at lm (3.020 mm, ruyjin, terumo, tokyo, japan) and lcx (2.520 mm, trek). After recovery of coronary flow, the patient's heart function seemed to improve echocardiography gradually . On the 6th hospital day, weaning from pcps was successfully, however, the patient was in septic condition, with decreasing courses due to unknown bacterial or fungal infections . Disseminated intravascular coagulation was getting worse and acute renal failure was not improving despite the continuous renal replacement therapy . Eventually, we lost the patient on the 15th hospital day . With the continued advances in percutaneous techniques for revascularization of coronary artery diseases, interventional cardiologist began to intervene in more complex coronary lesions . Entrapments on hardware materials of angioplasty have been reported for guidewires, balloons, rotablator systems, stents, and angiographic catheters usages.7)8) the complications of remnant guidewires could be life - threatening because it could lead to coronary thrombosis, perforation and embolization.3)4) bifurcation lesions, tortuous and calcified lesion, use of firm tipped guidewire are important risk factors for guidewire entrapments and fractures . Especially when the hydropilic coated guidewire tips can be easily broken in calcified and tortuous lesions . Therefore, the operator should more careful, when treating complex lesions with hydrophilic guidewires.9) there are several methods recommended for the treatment of fractured guidewire remnants . Some cardiologists with limited interventional experiences preferred urgent surgery . However, the surgery should be considered after failures of interventional methods, and there are no guidelines for surgical indications.9) percutaneous retrieval techniques of fractured guidewires include the use of snare loop or goose - neck snare, double or triple wire rotation,10) use of tornus catheter,11) use of a balloon as a wedge for extracting guidewire fragments12) and biopsy forceps.13) these techniques may be difficult and time - consuming for inexperienced cardiologists, and furthermore, they can cause endothelial injury, dissection and thrombosis.14) the best way to solve the sticky problem is the complete removal without complications . However, when it is not possible to get rid of the wires, fixing the thrombogenic materials to the vessel wall with stents can be a viable alternative to minimize the risk of thrombosis by device remant.9) remained guidewires in the coronary arteries may not always cause serious adverse effects . Non - metallic and hydrophilic guidewire tips are not highly thrombogenic, and it is reasonable to leave such remnants in small side branches or chronically occluded vessels.9) several conservative treatment cases were reported . In the reported cases, leaving the guidewire remnants within the left coronary artery and ascending aorta did not cause any serious complications during several months of follow - up period.5)6) conservative treatments are preferred when risks of interventional techniques or surgery outweigh its benefits in particular to patients who remain asymptomatic and hemodynamically stable.6) in our case, we have tried many ways from using snares to endoscopic biopsy forceps in order to remove the remnant wires . Despite all our efforts, upon closing of the procedure, we could remove only a part of the remnant in the ascending aorta . Because the patient refused further trials to remove the remnant wires, we had no choice but to leave the long and thin wire filaments into the proximal circumflex artery and distal lm where drug eluting stents had been deployed . Despite triple antiplatelet therapy, the result was thrombus formation within the stent at the lm . According to the case, we conclude that fractured guidewire is one of the most dangerous complications of pci . In similar cases, there are fears that the remnants within a coronary stent could result in stent thrombosis . If removal of such materials percutaneously is impossible, early surgical interventions should be considered.
Diabetic nephropathy (dn) represents an important cause of chronic kidney disease (ckd) that frequently leads to end stage renal disease (esrd). Diabetes mellitus (dm) is a frequent disease and dn is one of its main complications . It is appreciated that up to 40% of the patients with type i and type ii dm present dn . In western countries, diabetes is a leading cause of chronic kidney disease frequently leading to chronic renal replacement therapy (rrt) due to esrd . Taking into account the increased incidence of both dm and of dn, the detection of early dn is of paramount importance, in order to provide appropriate therapy that prevents or slows evolution towards esrd ., microalbuminuria represents a marker of the generalized endothelial dysfunction present in dm, linking renal involvement with cardiovascular and cerebral impairment . In time, it has been demonstrated that microalbuminuria reflects not only glomerular injury but also tubular lesions, filtered albumin being reabsorbed at tubular level . Additionally, new biomarkers have been studied in order to identify tubular lesions in dm . The new tubular biomarkers have been detected in both type 1 and type 2 dm early renal dysfunction that precedes microalbuminuria . At present, the assessment of early dn involves numerous biomarkers . They span the period of normoalbuminuria that precedes microalbuminuria but also the evolution of renal involvement during microalbuminuria and macroalbuminuria . Until they are universally accepted they are analyzed in relationship with the levels of albuminuria, especially of microalbuminuria . At present, markers of inflammatory and oxidative processes accompanying dm and dn are also being assessed . Since literature abounds in studies on markers highlighting renal dysfunction in different stages of the evolution of dm, we decided to restrict our study to the early phase of dn . An update of the urinary biomarkers used in early dn is useful for establishing their role in the early diagnosis of this disease, with subsequent prophylactic and therapeutic implications . We insist on urinary biomarkers because they are easily drawn, which allows population screening, and because they can detect tubular lesions, which occur very early in dm . Some of the biomarkers are constitutive elements of the nephron, such as markers atepithelial cell (podocyte) level, for example, nephrine and podocalyxin; glomerular basement membrane level: collagen and laminin; endothelial (vegf); tubular cell level, for example, ngal, nag, and kim . Epithelial cell (podocyte) level, for example, nephrine and podocalyxin; glomerular basement membrane level: collagen and laminin; endothelial (vegf); tubular cell level, for example, ngal, nag, and kim . Some have mixed origin; they can originate both in tubular cells and in podocytes, for example, angiotensinogen [7, 8]. Some are derived from the circulation, for example, transferrin, ceruloplasmin, and immunoglobulins g and m. they pass into the urine because of glomerular lesions which result in increased permeability for plasma proteins . Matheson classifies the biomarkers according to both their origin and the pathologic processes impairing the nephron: kidney damage, oxidative stress, and inflammation: biomarkers of renal dysfunction, inflammatory biomarkers (cytokines and chemokines),oxidative stress biomarkers . Biomarkers of renal dysfunction, inflammatory biomarkers (cytokines and chemokines), oxidative stress biomarkers . Another classification belongs to hong and chia who present 3 categories of biomarkers: glomerular, tubular, other proteins .it should be noted that products of metabolism in dm are also eliminated in the urine, and they can trigger toxic effects, for example, advanced glycation end products (age). Since we will frequently refer to microalbuminuria in presenting other biomarkers used in studying lesions of the nephron, namely, of the glomerulus and of the tubules, we will first present the main observations regarding microalbuminuria in diagnosing dn . Recent literature uses new terms, like moderately increased albuminuria for microalbuminuria and severely increased albuminuria for macroalbuminuria . However, the classical terms of microalbuminuria and microalbuminuria continue to be in wide use, as they are more practical . Kidney biopsy examination in patients with type 1 dm and microalbuminuria most frequently finds normal histological aspects . However, dn lesions were found in a small number of patients . According to mckenna and thompson, microalbuminuria is predictive element of the future development of end stage renal disease . Microalbuminuria can regress towards normoalbuminuria, it can persist as such, or it can progress towards albuminuria [14, 15]. The evolution of microalbuminuria towards macroalbuminuria is usually related to arterial hypertension and reduced gfr, an important part being generally played by risk factors . Persistent microalbuminuria is related to future development of end stage renal disease and to cardiovascular risk . It should be noted that diminution of gfr usually occurs after the development of microalbuminuria, but there are situations when even normoalbuminuria is accompanied by diminution of the gfr . Microalbuminuria is an important biomarker in type 2 dm, being frequently used in population - based screenings . Regarding the prevalence of microalbuminuria in type 2 dm patients we highlight a review of newman et al . They found microalbuminuria in 26% of the patients with ten - year duration of the disease . A study on 24,000 patients found that asian and hispanic patients with type 2 dm present more often microalbuminuria (43%) than whites (33%). In china, shanghai, microalbuminuria has a prevalence of 41% among patients with type 2 dm . It can regress towards normal values, it can progress towards macroalbuminuria, or it can remain unchanged . In a study on 216 patients, araki et al . Found, after a 6-year follow - up, regression of microalbuminuria in 51% cases and progression to severely increased albuminuria in 28% cases . The risk of progression to severely increased albuminuria is higher in patients with microalbuminuria as compared to patients with normoalbuminuria . The diminution of gfr is also higher in patients with severely increased albuminuria than in those with microalbuminuria . Glycemic control, ace inhibitors, and arbs for blood pressure control play an important role in the evolution of microalbuminuria . It should be mentioned that microalbuminuria has been considered a glomerular biomarker . To date, emerging data point to the role of the tubules in producing microalbuminuria [24, 25]. As such we did not include this marker among glomerular biomarkers but approached it separately, according to its potential role as both a glomerular and tubular biomarker . Transferrin is a protein with a molecular weight of 76.5 kda . Because of its low molecular weight and its less ionic load it filters easily through the glomerular barrier . As increased urinary transferrin was found in type 2 dm normoalbuminuric patients, concomitantly with urinary ceruloplasmin and immunoglobulin g, preceding microalbuminuria, it could be considered a biomarker of early dn . In microalbuminuric patients the levels of urinary transferrin increase . They also increase in patients with type 2 dm with vascular complications: coronary artery disease, diabetic retinopathy, and so forth . Patients with initial high levels of urinary transferrin excretion will develop microalbuminuria more frequently than those with normal levels . It is an anionic plasma protein with a molecular weight of 150 kda that crosses the glomerular barrier with difficulty . As presented above, urinary igg can be secreted before the stage of microalbuminuria, concomitantly with increased values of urinary transferrin, urinary ceruloplasmin, and urinary orosomucoid . It was also found in some type 2 dm patients with normoalbuminuria, arguing in favour of its use for early detection of renal lesions, even prior to albuminuria: ceruloplasmin could have in type 2 dm patients a dn predictive effect, similar to urinary transferrin and urinary immunoglobulin g . According to yamazaki et al . The urinary ceruloplasmin excretion rate (cer) and clearance of ceruloplasmin increase in parallel with the progression of albuminuria . In fact, in type 2 dm patients there could exist a parallelism between increased values of urinary transferrin, urinary immunoglobulin g, and urinary ceruloplasmin . Type iv collagen is a component of the glomerular basement membrane and of the mesangial matrix . In dn, lesions are produced both at glomerular capillary level and at mesangial level . Its excretion in urine might serve as early indicator of renal injury associated with dn . Increased levels of type iv urinary collagen are reported for normoalbuminuric patients with type 1 dm . Other authors also report increased excretion of type iv collagen and of laminin in patients with type 1 dm . High urinary type iv collagen excretion was also reported in normoalbuminuric patients with impaired glucose tolerance . Urinary type iv collagen could reflect morphological renal alterations in patients with type 2 dm . A relationship between the severity of histological lesions and urinary type iv collagen was reported in patients with type 2 dm . It could also allow both detection of early dn in patients with type 2 dm and differential diagnosis with glomerulonephritis, where its levels are low . Its urinary excretion is increased in normoalbuminuric type 2 dm patients, being correlated with nag (n - acetyl - beta - d - glucosaminidase) and alpha-1-microglobulin excretion . Glycosaminoglycans are components of the glomerular basement membrane as well as of the extracellular matrix . In dm alterations of these urinary glycosaminoglycans are associated with other tubular biomarkers, for example, tamm - horsfall protein, which expresses a distal tubular dysfunction in diabetic patients . It is a biomarker related to lesions of the glomerular capillary walls and reflects their increased permeability . It is mainly considered to predict renal lesions, being less relevant as an early marker of dn . These were studied only sporadically, without sufficient data to support their use as markers of early dn . Although the use of urinary glomerular biomarkers has not become current practice yet, glomerular biomarkers have been reported in some normoalbuminuric patients, leading to the conclusion that albuminuria might not represent the most sensitive glomerular biomarker . However, their clinical applicability needs to be confirmed in high - quality validation studies . Tubular biomarkers have shown that tubular dysfunction can be present early in dn, sometimes preceding glomerular injury . These biomarkers have proven highly sensitive as compared to microalbuminuria, which is considered the gold standard biomarker of dn . In fact, presently, microalbuminuria is regarded not only as a glomerular biomarker, but also as a tubular one . Neutrophil gelatinase - associated lipocalin is a glycoprotein present in the kidneys at tubular cell level and is considered to be protective against renal damage . Urinary ngal is a biomarker used in assessing tubular lesions in dm, its increased values being present even in the initial phases of the disease, namely, in normoalbuminuric patients . Thus, in type 1 dm high urinary ngal can precede microalbuminuria [50, 51]. Urinary ngal had high values in type 2 dm patients with normoalbuminuria, increasing progressively in patients with microalbuminuria and macroalbuminuria . The values of kim-1 (kidney injury molecule-1) increased in parallel, indicating an early and progressive lesion . However, fu et al . Reported in type 2 dm patients who present hyperfiltration and increased values of urinary ngal, as well as of urinary kim-1, as compared to the values of patients with normal gfr . Urinary ngal in type 2 dm patients could have a role in predicting the evolution of disease . Urinary alpha-1-microglobulin is a serum protein with low molecular weight (27-kda), which allows it to get easily filtered through the glomerular capillary wall . Tubular dysfunction leads to alteration of reabsorption with increased excretion in the urine . In a cross - sectional study, hong et al . Analyzed 590 type 2 dm patients and found that 33.6% patients with normoalbuminuria presented increased values of urinary alpha-1-microglobulin, a fact that could be explained by tubular injury that precedes the occurrence of microalbuminuria, being a more sensitive and an earlier urinary biomarker . This is why assessments of alpha-1-microglobulin are associated with the assessment of other urinary biomarkers, urinary albumin included . Reported high values of urinary alpha-1-microglobulin in normoalbuminuric patients, a fact pleading for an early tubular injury in type 2 dm in this stage . They did not find correlations between urinary alpha-1-microglobulin, beta-2 microglobulin, and the albumin / creatinine ratio with plasma asymmetric dimethyl - arginine . Reported in normoalbuminuric type 2 dm patients high values of urinary kim-1, which indicates lesions of the proximal tubule in early stages of the disease . De carvalho et al . Reported in type 2 dm normoalbuminuric patients high values of kim-1, these values increasing progressively in patients with microalbuminuria and macroalbuminuria . Moreover, kim-1 presents higher elimination in type 2 dm patients with hyperfiltration than in patients with normal glomerular filtration . These biomarkers kim-1 and ngal could plead for a deleterious lesional effect of hyperfiltration on the proximal tubule . . However could not demonstrate a value of urinary kim-1 that could be predictive of the evolution of glomerular function (gfr) in patients with type 1 dm . According to nielsen et al . Nag is an enzyme located in the lysosomes of proximal tubular cells . In case of dysfunction, namely, of injury of proximal tubular cells, nag is eliminated into the urine in higher quantities, being a sensitive tubular biomarker . Elevated serum cys c levels and urinary nag activities were found only in normoalbuminurics, not in controls ., did not find that urinary nag has clinical significance as an early biomarker of dn . In type 2 consider that urinary nag is the most sensitive biomarker for detecting early damage in diabetic patients . The renin angiotensin aldosterone system (raas) is involved in the pathogenesis of dn . The constitutive elements of raas are present at kidney level, defining a local raas . Urinary angiotensinogen in normoalbuminuric type 2 dm patients is higher than in controls and it increases progressively in microalbuminuric and especially in macroalbuminuric patients . Kim et al . Did not confirm these observations in a study on type 2 dm patients . They found that the values of urinary angiotensinogen are not different from those of the controls, in normoalbuminuric and microalbuminuric type 2 dm patients, but higher values were described in macroalbuminuric patients . These observations point to the need of further studies necessary for the validation of this biomarker . Increased urinary angiotensinogen could represent a risk factor in renal and cardiovascular complications . Since activation of raas could intervene in the evolution of dn, administration of ace - i is recommended . At the same time, urinary angiotensinogen could be a marker for assessing the renoprotective effects of alogliptin to type 2 dm patients . Assessment of gfr by means of cystatin c is considered to be a method that is not influenced by body mass, being comparable and even better than methods using serum creatinine . Patients with microalbuminuria present higher values of urinary cystatin c than those without microalbuminuria, urinary cystatin c having a predictive role for the progression of diabetic nephropathy (dn). Urinary cystatin c level could be an independent factor for identifying renal dysfunction in type 2 dm patients with normoalbuminuria, including patients with gfr <60/ml / min/1.73 m . Find a significant positive correlation between serum cystatin c, urinary nag, lacticodehydrogenase, alkaline phosphatase activities, and serum creatinine levels . Serum and urinary cystatin c are useful biomarkers for assessing early nephropathy in type 2 dm . Urinary l - fabp is a protein with low molecular weight expressed in the cytoplasm of human proximal tubular cells . Was found in type 1 dm patients who presented normoalbuminuria, having a predictive role regarding the progression towards microalbuminuria and of microalbuminuria towards macroalbuminuria . Patients with type 2 dm with normoalbuminuria also presented high levels of urinary l - fabp, this protein being considered as a useful biomarker for diagnosing early diabetic nephropathy . In fact, urinary l - fabp has been confirmed as a tubular biomarker by the ministry of health and welfare in japan . The values of urinary l - fabp increase with the decline of renal function . Although some authors, like chou et al ., do not ascribe a predictive role to urinary l - fabp in type 2 dm patients, others, like panduru et al ., consider that urinary l - fabp is an independent predictor of the progression of dn . Nephrine is a transmembrane protein in the structure of the slit diaphragm . In dm podocyte dysfunction nephrinuria was also reported in some normoalbuminuric type 2 dm patients [62, 90]. Dysregulation of nephrine in podocytes in dn could lead to nephrinuria in normoalbuminuric patients, preceding microalbuminuria . In albuminuric patients, nephrinuria is positively correlated with albuminuria and negatively correlated with gfr, being a biomarker of dn in other phases of dm as well . Podocyte impairment in dm involves not only nephrine but also other podocyte elements, for example, vegf . They manage to show, in most cases, that microalbuminuria does not represent a reliable biomarker for diagnosing incipient lesions of dn . However, up to now, none of these biomarkers has been established as gold standard for the identification of early dn . Dm is accompanied by chronic inflammatory processes affecting the whole body, the kidneys included . Urinary tnf alpha presents in type 2 dm patients with microalbuminuria and macroalbuminuria higher values than in patients with normoalbuminuria . Cherney et al . Analyzed in a complex study on normoalbuminuric type 1 dm patients forty - two urinary cytokines / chemokines . They found that the urinary level of il6 and il8, the platelet - derived growth factor, and rantes are not altered in patients with normal albumin - creatinine ratio . Consider that these markers could have a role in assessing the risk of dn in patients with type 1 dm . In type 1 dm patients, renal hyperfiltration is related to increased excretion of inflammatory cytokines / chemokines . Tashiro et al . Found in type 2 dm patients that il8 is high in early stages of dn and mcp-1 increases in advanced stages . A study on type 2 dm patients with normoalbuminuria and microalbuminuria found higher values of il8, ip10, mcp-1, g - csf, eotaxin, and rantes in patients with microalbuminuria than in normoalbuminurics or in controls . Ibrahim and rached also found that urinary mcp-1 is higher in patients with microalbuminuria than in normoalbuminurics or healthy controls . Urinary orosomucoid has higher values in type 1 dm patients with normoalbuminuria than in controls . Type 2 dm patients presented increased excretion of orosomucoid in the urine, in parallel with the excretion of immunoglobulin g, ceruloplasmin, and transferrin . Appreciate that orosomucoid is a significant independent factor for diabetic microvascular complications and can be considered as an early marker of renal injury . Urinary markers of inflammation are useful for assessing inflammatory processes in dn, even in early stages . It is eliminated into the urine without being metabolized . At present, it is considered a marker for oxidative stress . After a 5-year follow - up, hinokio et al . Find that 8-oxodg in urine is a useful clinical marker to predict the development of diabetic nephropathy in diabetic patients . There was a significant progression of diabetic nephropathy in the patients with higher excretion of 8-oxodg in urine compared with the patients with moderate or lower excretion of 8-oxodg . Reported increased excretion of 8-ohdg in type 1 dm patients 9 years after the onset of disease, mainly related to poor glycemic control . Heart fatty acid binding protein (h - fabp) is a marker of distal tubular damage . In a study on a cohort of type 1 and type 2 dm patients and an assessment of their markers of glomerular lesions (igg), markers of proximal tubular lesions (urinary kim-1, nag, ngal, and cystatin), and a marker of distal tubular lesions (urinary h - fabp) in relationship with albuminuria and gfr, nauta et al . Reported higher values of urinary nag, ngal, and h - fabp in normoalbuminurics than in controls . On the other hand, age eliminated in the urine induce a toxic tubular effect producing tubular dysfunction . In type 2 dm patients with normoalbuminuria high values of urinary alpha-1-microglobulin and of urinary kim-1 were found secondary to tubular dysfunction prior to the onset of microalbuminuria . At the same time, urinary age were high, being correlated with these markers . Found in type 2 dm patients high values of urinary age in 50% of the patients with normoalbuminuria and in 85% of those with microalbuminuria . Pentosidine, a component of age, is a biomarker for their formation and accumulation . Podocyte lesions appear during dm and dn, respectively, the disease being considered a podocytopathy as mentioned above . The assessment of podocyte injury can be accomplished by monitoring the number of podocyte cells in the urine or, more precisely, by means of using podocyte urinary biomarkers (podocalyxin and nephrine). A study on dm patients found that the values of the number of urinary podocytes in normoalbuminuric patients are not significantly different from those of controls . In patients with microalbuminuria and nephrotic syndrome, urinary podocalyxin originates in the podocyte apical surface, occurring in vesicle form . In dm patients, the podocalyxin level presented higher levels in patients with microalbuminuria than in patients with normoalbuminuria . Another study on dm patients found high values of urinary podocalyxin in more than half of the patients with normoalbuminuria, these values being higher in patients with microalbuminuria and macroalbuminuria . Urinary podocalyxin is correlated with the values of urinary nag and of urinary beta 2 microglobulin . Consider that urinary podocalyxin can be an early biomarker for detecting early podocyte injury in patients with dm . Zheng assessed the urinary microrna profile of podocyte - associated molecules (synaptopodin, podocalyxin, cd2-ap, -actin4, and podocin) as biomarkers in patients with normoalbuminuria, microalbuminuria, and macroalbuminuria and they reported its increase during the progression of dn . Urinary vegf was detected in type 2 dm patients, being correlated in these patients with urinary alpha-1-microglobulin, a biomarker for proximal tubular lesions . Kim et al . Found that vegf is excreted at higher values than controls in normoalbuminuric type 2 dm patients . Fetuin a is glycosylated glycoprotein was considered an inhibitor for ectopic calcium deposition and promoter of insulin resistance . It was found that elevated urinary fetuin a excretion is a risk for development of diabetic nephropathy . Urinary retinol - binding protein is a low molecular weight protein that was found to have high urinary values (together with nag) in normoalbuminuric patients, reflecting tubular dysfunction in early dn . The value of serum retinol - binding protein 4 as a biomarker in assessing the severity of coronary artery disease is to be mentioned . Urinary retinol - binding protein 4 as a biomarker in assessing dn needs further studies . Urinary heme oxygenase-1 was found in type 2 dm patients before the onset of significant albuminuria, thus being a possible biomarker of early dn . Periostin is a cell adhesion molecule which is not normally present in kidneys . In tubulointerstitial lesions it is however expressed in the kidneys, being eliminated in the urine . This is why urinary periostin could be used as a marker of injury at this level . Since high levels of periostin can be identified in dm patients before significant albuminuria, periostin could represent a marker of diabetic renal injury . Urinary alpha klotho presents higher values in normoalbuminuric type 2 dm patients than in controls . It can also be a marker of diabetic injury . Analyzing a group of normoalbuminuric, microalbuminuric, and macroalbuminuric type 2 dm patients, sun et al . Noted that the urinary level of microvesicle - bound dipeptidyl peptidase - iv is related to the severity of dn . Recent studies point to the usage of urine - specific microrna as a biomarker for early stages of dn . Analyzing the studies in the literature, yang et al . Issued the hypothesis that urine - specific microrna would be a marker that can be used in the early stages of dn . Recently, argyropoulos highlighted the predictive role of urinary microrna regarding microalbuminuria in type 1 dm . Adipokine zinc - alpha-2 glycoprotein is assigned to the major histocompatibility complex class i of proteins . Also appreciate adipokine zinc - alpha-2 glycoprotein as a novel urinary biomarker for normoalbuminuric diabetic nephropathy . At present proteomic investigations are engaged in identification of new urinary biomarkers to be used in the early diagnosis of dn . In fact, proteomics studies noted the fact that microalbuminuria is not a perfect biomarker for early detection of dn [130, 131]. Urinary proteomics begins to stand out as a noninvasive method of detecting early dn . Among proteomics studies on diagnosing dn, who reported that collagen fragments were a prominent biomarker 35 years before the onset of microalbuminuria . A potential role is also attributed to exosome proteomics for identifying new biomarkers for dn . Showed a panel of 3 proteins which is differentially present in urinary exosomes from dn patients . Urinary proteomic analysis can have an important role in the implementation of new biomarkers in dn . At present, the prospect of discovering new biomarkers in dm and dn respectively is incumbent both on proteomics and on genomics, transcriptomics, and metabolomics . Microalbuminuria, although frequently contested as a biomarker of early dn, is used so far as reference biomarker in assessing other urinary biomarkers in early dn . Until present there is no other biomarker that can substitute in practice microalbuminuria, the new biomarkers being sustained by limited studies and requiring validation . The concomitant assessment of several urinary biomarkers in relationship with microalbuminuria could represent at present a method of diagnosing early dn . The great progress in discovering new biomarkers could lead to the development of an ideal urinary biomarker to detect early diabetic dn in the future . Progresses in the field of urinary biomarkers in dn, promising both in proteomics and in other modern techniques, develop remarkably at present.
A traditional individual - based ethics would say that one should not exaggerate but this cannot capture the whole story of ambivalence in promising technology . A technology such as nanotechnology needs promises, including some exaggeration, to persuade target audiences . Thus, actors have to participate in the strategic game of promising (sometimes beyond reason). Current developments in the world of nanotechnology demonstrate the role of position and context and strategic games in promising technology . There is a back - and - forth pattern of ethical argumentation between proponents and opponents of the technology . The arguments put forward by the actors are indicative of their view of what the some stable distribution of responsibilities (and thus, a division of moral labour) may evolve . This distribution of responsibilities may well be justified and productive but it is primarily the outcome of a struggle, a larger and more distributed version of the struggle that latour illustrated with the program and anti - program of the hotel manager and the hotel guests in relation to the hotel key . Such positioning and the patterns that are involved have been analysed before; i will offer a brief overview in the next section . What is new for nanotechnology is that such discussions and positioning have become pervasive and that actors have to be articulate about them . Industrial actors are interesting in this respect, because they are closer to actual applications and their repercussions than scientists . This was part of the ethics in the real world workpackage in the eu - funded deepen (deepening ethical engagement and participation in emerging nanotechnologies) project.1 the industrial actors struggled with ambivalences in nanotechnology; their responses to these ambivalences were linked with their position as industrial actors and the particular contexts within which they act . Interestingly, and indicative for the new situation around nanotechnology, their interactions with ngos influenced how they dealt with ambivalence in nanotechnology . Enactor perspective; the promise of nanotechnology must be pushed and ethics is seen as a brake on progress . However, they can be more nuanced, as our interview data show . By way of a preface, one vignette describes the strategic considerations towards hype followed by biotechnologists in the netherlands in the 1980s, while the other recapitulates the use of promises about embryo research by opponents of the research . Proponents of a technology are faced with strategic considerations with regard to whether they want to contribute to hype around a technology or be more reasonable in referring to the promise of a technology; these strategies can be combined with different resource mobilisation strategies . Rip and nederhof observed that these two strategies were pursued by scientific researchers in the netherlands in the 1980s, in the move towards biotechnology - relevant research . Biochemists and molecular biologists made strong claims especially because they were more removed from actual practices of biotechnology than microbiologists and chemical engineers, who were more modest . As biotechnology was not an essential component of their ongoing research, biochemists and molecular biologists were re - labelling (, 258) their ongoing research, so that they too could join the biotechnology band - wagon and avail of policy funding opportunities . The promises of proponents of a technology can be strategically used by opponents to undermine their credibility . Mulkay examines some of the rhetorical resources used by opponents and proponents of embryo research during a parliamentary debate in the uk house of commons in 1990 . Look into the future and focus on the expected outcomes of scientific research (, 728). Proponents of the research used the rhetoric of hope to make strong claims about the promise of the research, even though the details of the development were as yet unknown . For opponents, the tangible achievements of embryo research to date were judged to be negligible and the rhetoric of fear was used to challenge opponents claims, transforming them into a collection of misleading exaggerations (, 730). These vignettes demonstrate actors response to the ambivalence of hype and, in turn, their response to the opportunities of a promising technology (in the first case) and the concerns evoked by a promising technology (in the second case). To continue this discussion, the ethics of promising technology suggests that one should not exaggerate without reason but also that there may well be reasons: one has to mobilize resources to be able to realize (materialize) the promises, and has to do so in competition with many other claims on such resources (, 18). One has to claim more than is reasonable, in order to be able to realize what is actually a reasonable claim (, 18). It leads to two different actor strategies: one can choose to sustain the hype or be more modest in presenting the promise of a technology . While there is an immediate opportunistic argument to pursue the hype strategy, there are two arguments against it . As a result of inflated promises, research investment may be directed into unfeasible areas of research, to the detriment of other research communities that could have benefited more from investment, resulting in wasted resources and missed opportunities . Sparrow and swierstra and rip argue that proponents of nanotechnology often try to have it both ways in arguments about the nature and impact of nanotechnologies, describing nanotechnologies as both revolutionary and evolutionary, depending on the discourse surrounding nanotechnologies at the time: in arguments about their nature and impact we are simultaneously informed that these are revolutionary technologies with the potential to profoundly change the world and that they merely represent the extension of existing technologies (, 57). This ambivalence is visible in patterns of ethical argumentation about new and emerging science and technology described by swierstra and rip . The pattern starts with promises voiced by proponents, claiming major changes, all for the good of mankind, that is, the technology is revolutionary. In response, opponents of the technology who are calling for a cautious approach highlight the novelty of the new technology in order to bring attention to the dearth of knowledge about effects of the new technology . The proponents then face a problem; they had initiated the discussion by stressing the novelty of the technology in order to attract allies and mobilise resources . They are now forced to downplay the novelty of the emerging technology and present it as nothing unusual; what was once termed this downplaying of novelty by proponents of the new technology is just one move in the strategic game played between proponents and opponents . Revolutionary nanotechnology is threatened by opponents concerns, the proponents are forced to present nanotechnology as evolutionary nanotechnology is deployed according to the need to hype up or play down nanotechnology in response to opposing voices, it is clear that this ambivalence depends on the particular context in which nanotechnology is discussed . This form of context - dependence can also manifest itself in the specific qualification of evolutionary. In an interaction between a prominent representative of industry and a leading ngo representative at a seminar on policy - making in nanotechnology, i observed how the notion of the industry representative began her presentation by saying that nanotechnology should be seen as a revolution in quality of life rather than as an industrial revolution . Revolutionary nanotechnology as the next industrial revolution which will profoundly change the world for the better . In correspondence following the meeting, the industry representative told me that the message she wanted to convey was of the great impact of nanotechnology rather than that of an industrial revolution because of the specific view of industrial revolution, which is not altogether positive; thus it seemed that the industry representative was cognisant of the need to tailor the notion of, the opposing discussant was an important factor and the industry representative demonstrated awareness of what kinds of argument might appeal to or even this argument is most often used by commentators and critical groups who will sometimes describe a good life and use this as a reference in discussions about a promising technology; on the other hand, proponents of a promising technology often push the promises and fantastic possibilities of a new technology without reflecting on what kind of nanotechnology is (emphasis added) a major industrial revolution. In this interaction, it appears that the ngo representative is also aware of the use of qualifying major industrial revolution enabled him to use the negative connotation of industrial revolution in a counter strategy to argue for a moratorium on nanotechnology r&d . These are general considerations . How do industrial actors manage these ambivalences and tensions when faced with strong claims about the promises of nanotechnology emanating from policy makers and being taken up in society, as well as the emerging discourse about responsible development of nanoscience and nanotechnologies and possible restrictions on unfettered development of nanotechnologies? We mapped the ethical commitments and patterns of moral argumentation of industrial actors whose company had some involvement in nanotechnology . The respondents came from multinational companies including a chemical company, two semiconductor companies, a beverage and foodstuffs company and a big conglomerate including a food company . In some sectors, nanotechnology is an enabling technology which delivers new materials and components to help create better devices and systems which provide desired functionalities . In other sectors, nanotechnology just improves performance and sometimes allows new functionalities (e.g. Surfaces that repel dirt) but the constitutional effects derive from the system and how it is embedded and used . For example, when rfid (radio frequency identification devices) becomes cheaper and smaller, thanks to nanotechnology, and thus more widely usable and easier to implant, all products can be traced individually and an internet of things becomes possible, leading to a view of the implantable and thus readable human . All this is yet to come but it is being discussed already and may lead to measures and arrangements . For new materials, it is indicative that chemical firms have developed nanotechnology codes of conduct . Responsible care program) and think they can meet the credibility pressures . For other domains under the umbrella of nanotechnology, revolutionary. We began the interviews by asking the respondents about their company s involvement in nanotechnology and their stance towards responsible innovation in nanotechnology.3 they described nanotechnology as an evolution in their development and responsible innovation in nanotechnology as a normal part of their corporate social responsibility, thus business as usual. This description of development in nanotechnology as business as usual was evident in the bemused response of a respondent from a chemical company when asked how his company came to be involved in nanotechnology . He replied we are a chemical company, so why the question? He went on to describe nanotechnology as a natural step in development when nanotechnologies are defined as the next step to control materials at an ever smaller scale. In another version of business as usual, the respondent from the beverage and foodstuffs company explained that there is already nanotechnology which is not new in the food chain . One respondent explained that nanotechnology simply represents a solution to a technical problem . In the semiconductor sector, micro - electronics includes more and more nanotechnology in its miniaturisation drive . Thus, for this sector, nanotechnology is not revolutionary; it is the continuation of microtechnology . The industrialists use of the nothing unusual argument is general but is articulated differently . The response of the respondents from the chemical company and the beverage and foodstuffs company highlights that there is nothing yet at stake for the companies in terms of having to deal with specific nanotechnology - related issues such as ethical, environmental and health and safety issues . Their response emphasised downplaying the promise of nanotechnology; these sectors of industry are aware that nanotechnology evokes certain fears and concerns so they are careful to stress that nanotechnology is nothing new and thus not cause for concern . The semiconductor sector, on the other hand, does not see these concerns as relevant for them, if they are aware of them at all . When we asked the respondents for their view on calls for a ban on nanotechnology development4 nanotechnology became revolutionarywith the exception of the respondents from the semiconductor companies and could contribute to efforts towards climate protection or to the fight against cancer . This was then linked to an ethical argument against a moratorium on nanotechnology development.5 i offer a few quotes to support this diagnosis:nanotechnology would be good for the environment, for energy use, etc . And if you look at nanomedicine what they talk about nobody can be against it...if you design a medicine in such a way that it finds the right place to be released in the body without any additional side - effect (respondent from the chemical company). Nanotechnology would be good for the environment, for energy use, etc . And if you look at nanomedicine what they talk about nobody can be against it...if you design a medicine in such a way that it finds the right place to be released in the body without any additional side - effect (respondent from the chemical company). The benefits of nanotechnology in the push towards climate protection were noted by the respondent from a big conglomerate including a food company who felt that a moratorium on nanotechnology r&d would slow down the development of solar cells...when everybody outside is saying, the single biggest threat to our human species is climate change.... he added [the] same applies for preservation of foods in hunger stricken areas. This respondent continued by making claims of ethically responsible action and said that he considered a moratorium on commercial development of nanotechnology to be itself possibly ethically questionable. In the last quotes, the nothing unusual argument is inverted; the proponents of the new technology stress the novelty of nanotechnology when defending continuation of its development . This differs from the pattern of moral argumentation described by swierstra and rip . In this case proponents of nanotechnology adopt the strategies of opponents to highlight the novelty of nanotechnology but for a different cause than that pushed by opponents . Here the novelty argument or revolutionary argument is used in order to defend development of nanotechnology . Thus the respondents use the revolutionary argument when there is something at stake for them as proponents of nanotechnology; in this case it is their right to continue to work on nanotechnology development . There was a clear shift in focus from the technical or the performance aspect when referring to the evolutionary nature of nanotechnology to the social problem / solution dichotomy when pronouncing on the revolutionary potential of nanotechnology . When respondents referred to social issues such as medical treatments and climate change, which are both high on the agenda in discussions in the social sphere, nanotechnology became revolutionary . Positioning nanotechnology as a potential solution to social problems facilitated the call for ongoing development of nanotechnology . They realise that it is in their interest to present nanotechnology as business as usual or evolutionary in order to render nanotechnology familiar and therefore harmless . On the other hand, they offer the promise of such an argument assumes that their audience will want progress (through nanotechnology) and so will be receptive to their claim . Ngos and civil society organisations may, or may not, go along with this . An example is the friends of the earth report on agricultural and food applications;6 while foe wants improvement of agriculture, the nano route will prejudice precision agriculture as an alternative to the biological / ecological route favoured by foe . The ethical arguments of the industrial actors referred to their notion of progress through the development of nanotechnology . Indeed their response to the ambivalence of the nature and impact of nanotechnology was predicated on their need to be allowed to continue with the development of nanotechnology . This argument emerged when they made claims of ethically responsible action, for instance, the development of organic solar cells to be deployed in the fight against climate change . Thus responsibility was explicitly framed in terms of the development of nanotechnology to meet societal challenges . This then allowed them to turn the stance of ngos that industry is ethically questionable in its pursuit of nanotechnology back onto the ngos; if ngos stop development in nanotechnology, then they are the ones who are unethical . The ethical arguments which the industrial actors used to justify development in nanotechnology reflect standard repertoires in which scenarios of promise are pushed and ethics is viewed as a brake on progress . Division of moral labour refers to a division of obligations and commitments, as well as to notions regarding who is eligible to be praised or blamed . The present division of moral labour creates a space in which scientists and other technology developers, such as industrial actors, can focus on the progress of science and technology, while other actors (government agencies, ngos) are expected to look after other considerations, including ethical and social ones . Indeed the industrial actors view of the role of ngos in the responsible development of nanotechnology referred to a division of moral labour . The industrial actors felt that ngos had the right to ask critical questions and indeed, that somebody should ask questions on behalf of the public . This was reflected in one respondent s view that ngos are entitled to their position even when they call for a moratorium on nanotechnology development . Another respondent felt that ngos concern about nanotechnology is...a very good thing, in the sense that there are groups of people who watch the developments and look critically at them, ask questions to make sure that everybody is keen on the balance between opportunities and the potential risks . Well, that s the impression i have . Also, i believe that even the groups that are sort of aware and ask critical questions, my personal impression is that they are also looking for the balance about what is really the issue and only the ones who are very political will make a firm statement like there should be a ban before we know enough....a very good thing, in the sense that there are groups of people who watch the developments and look critically at them, ask questions to make sure that everybody is keen on the balance between opportunities and the potential risks . . Also, i believe that even the groups that are sort of aware and ask critical questions, my personal impression is that they are also looking for the balance about what is really the issue and only the ones who are very political will make a firm statement like there should be a ban before we know enough. Here the respondent introduces a distinction between this is an enlightened view; there are also industrial actors who are furious over the activities of ngos . In our interviews, we heard respondents accuse ngos of being agitators, failing to act in good faith, using misleading information to further their cause and painting different nanotechnologies with the same brush . A division of moral labour is effective when it is accepted and implemented, that is, when it is solid. However, in changing circumstances (which might include changing values, e.g. About precaution or about participation), where responsibilities may have to be redefined, the solidity of the division of labour will become a hindrance rather than a help . It has to be opened up or melted down to allow space for new configurations . While the industrial actors views about ngos reinforced this division of labour, new configurations in the world of nanotechnology, such as the collaboration between the chemical manufacturing company dupont and the non - profit group environmental defense, suggest that there is already a move towards redefining responsibilities in the world of nanotechnology . In 2005, dupont and environmental defense (who could be viewed as adversarial stakeholders) formed a partnership to work together to produce a nano risk framework,7 aimed at evaluating and addressing potential environmental, health and safety risks of nanomaterials across the entire life cycle of the materials . Dupont evaluated the framework deemed appropriate by both organisations using three case studies and published the results in early 2007 . Both organisations have issued a call for feedback on the framework and have encouraged companies to adopt the framework . Interestingly, there was a response from a civil society labour coalition,8 which issued a statement condemning what they called the the corporate partnership between dupont and environmental defense may set a precedent for a new kind of interaction between industry and ngos . On the other hand, the strong response from the civil - society labour coalition reasserted the traditional boundaries and division of moral labour which exist between industry and civil society organisations . At this stage, it is impossible to decide whether the civil - society labour coalition s reaction is conservative in the face of ongoing overall changes, or an indication that such changes are not occurring, and the dupont - environmental defense collaboration is a passing occurrence . Responsible development can, and will, be contested because changes in division of moral labour are about politics just as much as they are about ethics . This is visible in my interview data as well, even while there is no action involved . The ambivalences of nanotechnology, and of promising technologies more generally, are not just an intellectual challenge; the evolving strategic games that are played are for real, even if the discourse of responsible development may start as a new language game.
Toxicological risk assessment has historically relied on the use of animal models which, though fundamental in the life sciences, are also linked with shortcomings such as inconsistent translatability to humans and high cost . Furthermore, there has been an increasing effort to find alternatives to animal testing in the spirit of " the 3rs " (replacement, reduction, and refinement). This effort has been accelerated over the past few years, not only because of recent advances such as high - throughput techniques and systems biology approaches, but also because of legislation restricting the use of animal testing, especially in the european union . The complexity of cellular signaling pathways regulating the response to toxic insults makes it evident that using single toxicological endpoints will not be sufficient to describe the toxicological basis of certain compounds . For this, the interplay of hundreds of interacting proteins contributing to a biological network will also need to be taken into account . To study the effect of toxicants on those networks, a system toxicology approach combined with phenotypic medium- and high - throughput screening assays is useful to infer potencies and at the same time provide more information on the mechanism of action of individual toxicants . In this study, we employed hcs as a powerful screening tool, which is composed of an automated microscope and a biological software application, that can acquire, process and analyze image data derived from specific fluorescence - based cellular assays . This allows for visual changes within a cell to be quantified, at a single cell or subcellular level, and many parameters to be analyzed simultaneously . For example, dna double - strand breaks were evaluated using an antibody - based identification of histone h2ax phosphorylation and reactive oxygen species (ros) were quantified using a cell - permeable superoxide sensitive dye . Because lung epithelial cells represent the first biological barrier against inhaled toxicants, including cigarette smoke, we utilized primary bronchial epithelial cells as an in vitro model to profile the effect of hphcs published by the united states food and drug administration . This manuscript is a follow - up on a previous study in which we evaluated the biological impact of a different subset of hphcs . As part of our workflow to assess cytotoxicity in vitro, we initially evaluated the potencies of a selection of 15 hphc's, using an impedance - based real - time cellular analysis (rtca) system which allowed us to establish dose - ranges, suitable for subsequent hcs analysis (figure 1). A toxicological hcs assessment was then conducted using nine multi - parametric endpoints of cellular toxicity, each monitored at two time points (4 and 24 hr). The markers used were indicative of mitochondrial toxicity, dna damage, stress kinase, reactive oxygen species (ros), glutathione (gsh) content, caspase 3 - 7 activity, cytochrome c release and cell membrane permeability, as described in table 1 . Our approach enabled identification and characterization of the effect of cigarette smoke constituents through dose- and time - dependent sampling . This would finally also provide a deeper understanding of the effects at the cell signaling and/or transcriptional level . Pre - warm the cell culture medium (bronchial epithelial cell growth medium - supplemented medium), the hepes, trypsin, and trypsin neutralizing solution (tns) in the water bath at 37 c . Note: the following nhbe cell culture seeding conditions may be used to obtain optimal confluence in uncoated t75 flasks with 20 ml medium: seed 1 x 10 cells for 3-day culture, 0.5 x 10 cells for 4-day culture and 0.25 x 10 cells for 5-day culture . Change medium every 2 days when cells are in culture to refresh nutrients . Seed 1 x 10 cells for 3-day culture, 0.5 x 10 cells for 4-day culture and 0.25 x 10 cells for 5-day culture . Remove the supernatant from the flask(s) and add hepes to wash the cells (e.g., 3 ml for a 75 cm flask). Remove the hepes solution and add trypsin solution (e.g., 3 ml for a 75 cm flask). Rotate the flask to cover the cell monolayer with trypsin solution . Monitor cell detachment under the microscope and if necessary, incubate longer and gently tap the flask to release any remaining attached cells . Add tns to stop the reaction (e.g., 3 ml for a 75 cm flask) and transfer the cell suspension to a 15 ml tube . Discard the supernatant and re - suspend the cell pellet in 10 ml of fresh medium, mixing gently to yield a homogeneous cell suspension . Filter the cell suspension through a 100 m cell strainer to remove aggregates and count the cells . Note: in our laboratory an electric field multi - channel cell counting system was used to precisely and consistently evaluate the viable cells number . Note: an impedance - based measurement system was used to: 1) evaluate compound toxicity, 2) select compounds to be further investigated by hcs and 3) select appropriate doses for hcs . The nhbe cells in the rcta plates are dosed by adding 25 l of test compound dilutions to 100 l medium present in each well . Therefore, all test solutions are prepared at 5 times (5x) the desired final concentration . Seeding nhbe cells program the instrument to define the number and duration of impedance measurements . In this study, data were recorded every 15 min for 48 hr (from 24 hr 2 hr before and 24 hr after dosing the cells with test agents).measure the plate background by pipetting 50 l of pre - warmed medium into each well of a 96-well rtca plate . Note: this step represents a technical requirement for the instrument to calculate the medium electrical resistance which is then used as a baseline reference for cell - based calculation.prepare a cell suspension at a concentration of 144,000 cells / ml (5%) and add 50 l cell suspension (7,200 cells / well) to each well of the rtca plate in which 50 l / well of medium was already added for instrument background measurement.let the cells adhere for 30 min at room temperature before placing them into the rtca cradle (to improve the homogeneous distribution of the cells). Incubate the rtca plates in the rtca cradle in the incubator (37 c and 5% co2) and start recording the data for the next 24 2 hr prior to dosing . Program the instrument to define the number and duration of impedance measurements . In this study, data were recorded every 15 min for 48 hr (from 24 hr 2 hr before and 24 hr after dosing the cells with test agents). Measure the plate background by pipetting 50 l of pre - warmed medium into each well of a 96-well rtca plate . Note: this step represents a technical requirement for the instrument to calculate the medium electrical resistance which is then used as a baseline reference for cell - based calculation . Prepare a cell suspension at a concentration of 144,000 cells / ml (5%) and add 50 l cell suspension (7,200 cells / well) to each well of the rtca plate in which 50 l / well of medium was already added for instrument background measurement . Let the cells adhere for 30 min at room temperature before placing them into the rtca cradle (to improve the homogeneous distribution of the cells). Incubate the rtca plates in the rtca cradle in the incubator (37 c and 5% co2) and start recording the data for the next 24 2 hr prior to dosing . Dilutions of hphcs and positive controls positive control dilution dilute the staurosporine stock solution (10 mm) 1:10 in dmso (see table 3) and add 5 l of the dilution to 195 l of medium to obtain a 5x working solution . Hphc dilution dissolve / dilute each hphc in the vehicle (table 2) to generate a 1 m stock solution . Dilute each hphc stock solution 1:10 in medium to generate a 100 mm solution.generate the " compound master plate " by performing a five - step 1:10 serial dilution using medium + 10% vehicle to obtain the 5x working solutions (figure 2). Note: ultimately, the final doses will be: 0.2 m, 2 m, 20 m, 0.2 mm, 2 mm, 20 mm . Also prepare the dose 0, corresponding to the only vehicle, in this step . Dosing pause the rtca instrument and open the cradle to remove the plate.remove the plate and place it in the rtca plate temperature tool (designed to stabilize the temperature of the rtca plate during experimental procedures outside the rtca station) to avoid cooling the cells, which could impact the impedance measurement.add 25 l 5x solution from the " compound master plate " to cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 2). Do not remove the existing (100 l) cell culture medium.add 25 l 5x positive control solution to the cells in the bottom row (half - right) without removing existing culture medium . Add 25 l medium to the cells in the bottom row (half - left) without removing existing cell culture medium.seal the plate with plate sealer . Note: the use of this sealant film is recommended to avoid potential contamination, including well - to - well cross - contamination . Data analysis rtca and ld50 calculation export raw data as a text (.txt) or excel (.xls) file . Note: the file will contain all the information regarding the plate layout (compounds, doses and well position). Raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every time points at which the recording occurred . The value at position i in the 96 well plate at time t is denoted by cit(i).identify as a normalization reference the latest time point before the dosing for each position i in the 96 well plate (e.g., cell index at 23 hr 50 min 00 sec at position i, cit(i)=23:50:00 = ciref(i)). Note: this information must be annotated when the dosing is performed.divide, on a well base, every time point values by the normalization reference to normalize all the values at the dosing time . The normalized value at position i in the 96 well plate at time t is denoted by ncit(i) and is thus defined by ncit(i) = cit(i) / ciref(i) for all t.calculate the area under the curve (auc) at 24 hr post - dosing for each sample i (position i in the 96 well plate), including positions for positive control and vehicle . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values normalize the values using the following equation: where i is the position in the 96 well plate for which auc was calculated at 24 hr post - dosing, vehicle is the median of the auc values for the vehicle wells on a plate at 24 hr post - dosing, positive control is the median of the auc values for the positive control wells on a plate at 24 hr post - dosing, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (-100%) note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that is applied to the sample contained in position / well i and its corresponding normalized auc at 24 hr post - dosing auc_normalized(i).plot and fit the (ci, auc_normalized(i))-values using a 4-parameters hill equation . Where y = auc_normalized, zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Positive control dilution dilute the staurosporine stock solution (10 mm) 1:10 in dmso (see table 3) and add 5 l of the dilution to 195 l of medium to obtain a 5x working solution . Dilute the staurosporine stock solution (10 mm) 1:10 in dmso (see table 3) and add 5 l of the dilution to 195 l of medium to obtain a 5x working solution . Hphc dilution dissolve / dilute each hphc in the vehicle (table 2) to generate a 1 m stock solution . Dilute each hphc stock solution 1:10 in medium to generate a 100 mm solution.generate the " compound master plate " by performing a five - step 1:10 serial dilution using medium + 10% vehicle to obtain the 5x working solutions (figure 2). Note: ultimately, the final doses will be: 0.2 m, 2 m, 20 m, 0.2 mm, 2 mm, 20 mm . Also prepare the dose 0, corresponding to the only vehicle, in this step . Dissolve / dilute each hphc in the vehicle (table 2) to generate a 1 m stock solution . Generate the " compound master plate " by performing a five - step 1:10 serial dilution using medium + 10% vehicle to obtain the 5x working solutions (figure 2). Note: ultimately, the final doses will be: 0.2 m, 2 m, 20 m, 0.2 mm, 2 mm, 20 mm . Also prepare the dose 0, corresponding to the only vehicle, in this step . Dosing pause the rtca instrument and open the cradle to remove the plate.remove the plate and place it in the rtca plate temperature tool (designed to stabilize the temperature of the rtca plate during experimental procedures outside the rtca station) to avoid cooling the cells, which could impact the impedance measurement.add 25 l 5x solution from the " compound master plate " to cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 2). Do not remove the existing (100 l) cell culture medium.add 25 l 5x positive control solution to the cells in the bottom row (half - right) without removing existing culture medium . Add 25 l medium to the cells in the bottom row (half - left) without removing existing cell culture medium.seal the plate with plate sealer . Note: the use of this sealant film is recommended to avoid potential contamination, including well - to - well cross - contamination . Remove the plate and place it in the rtca plate temperature tool (designed to stabilize the temperature of the rtca plate during experimental procedures outside the rtca station) to avoid cooling the cells, which could impact the impedance measurement . Add 25 l 5x solution from the " compound master plate " to cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 2). Add 25 l 5x positive control solution to the cells in the bottom row (half - right) without removing existing culture medium . Add 25 l medium to the cells in the bottom row (half - left) without removing existing cell culture medium . Note: the use of this sealant film is recommended to avoid potential contamination, including well - to - well cross - contamination . Data analysis rtca and ld50 calculation export raw data as a text (.txt) or excel (.xls) file . Note: the file will contain all the information regarding the plate layout (compounds, doses and well position). Raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every time points at which the recording occurred . The value at position i in the 96 well plate at time t is denoted by cit(i).identify as a normalization reference the latest time point before the dosing for each position i in the 96 well plate (e.g., cell index at 23 hr 50 min 00 sec at position i, cit(i)=23:50:00 = ciref(i)). Note: this information must be annotated when the dosing is performed.divide, on a well base, every time point values by the normalization reference to normalize all the values at the dosing time . The normalized value at position i in the 96 well plate at time t is denoted by ncit(i) and is thus defined by ncit(i) = cit(i) / ciref(i) for all t.calculate the area under the curve (auc) at 24 hr post - dosing for each sample i (position i in the 96 well plate), including positions for positive control and vehicle . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Normalize the values using the following equation: where i is the position in the 96 well plate for which auc was calculated at 24 hr post - dosing, vehicle is the median of the auc values for the vehicle wells on a plate at 24 hr post - dosing, positive control is the median of the auc values for the positive control wells on a plate at 24 hr post - dosing, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (-100%) note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that is applied to the sample contained in position / well i and its corresponding normalized auc at 24 hr post - dosing auc_normalized(i).plot and fit the (ci, auc_normalized(i))-values using a 4-parameters hill equation . Where y = auc_normalized, zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Export raw data as a text (.txt) or excel (.xls) file . Note: the file will contain all the information regarding the plate layout (compounds, doses and well position). Raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every time points at which the recording occurred . The value at position i in the 96 well plate at time t is denoted by cit(i). Identify as a normalization reference the latest time point before the dosing for each position i in the 96 well plate (e.g., cell index at 23 hr 50 min 00 sec at position i, cit(i)=23:50:00 = ciref(i)). Divide, on a well base, every time point values by the normalization reference to normalize all the values at the dosing time . The normalized value at position i in the 96 well plate at time t is denoted by ncit(i) and is thus defined by ncit(i) = cit(i) / ciref(i) for all t. calculate the area under the curve (auc) at 24 hr post - dosing for each sample i (position i in the 96 well plate), including positions for positive control and vehicle . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Normalize the values using the following equation: where i is the position in the 96 well plate for which auc was calculated at 24 hr post - dosing, vehicle is the median of the auc values for the vehicle wells on a plate at 24 hr post - dosing, positive control is the median of the auc values for the positive control wells on a plate at 24 hr post - dosing, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (-100%) note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that is applied to the sample contained in position / well i and its corresponding normalized auc at 24 hr post - dosing auc_normalized(i). Where y = auc_normalized, zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Note: a total of nine multi - parametric markers of toxicity, grouped in six different assays, are measured using the hcs platform (table 1). Based on the rtca cell viability analysis (section 2) the dose range of each constituent is defined and a 3r4f reference dose is also included . The reference dose is equivalent to the amount of hphc present in the smoke of one stick from the reference cigarette 3r4f . Seeding nhbe cells prepare a cell suspension at 120,000 cells / ml (5%) and add 100 l cell suspension to each well of a 96-well hcs plate (12,000 cells / well). Prepare enough plates for the assessment of all assays (cytotoxicity, dna damage, stress kinase, ros, gsh content and apoptosis) and timepoints (4 and 24 hr).leave the hcs plates at room temperature for 30 min to allow cells to attach to wells and then incubate at 37 c and 5% co2 for 24 2 hr prior to dosing . Prepare a cell suspension at 120,000 cells / ml (5%) and add 100 l cell suspension to each well of a 96-well hcs plate (12,000 cells / well). Prepare enough plates for the assessment of all assays (cytotoxicity, dna damage, stress kinase, ros, gsh content and apoptosis) and timepoints (4 and 24 hr). Leave the hcs plates at room temperature for 30 min to allow cells to attach to wells and then incubate at 37 c and 5% co2 for 24 2 hr prior to dosing . Dilution of hchps and positive controls note: the nhbe cells in the hcs plates will be dosed by adding 25 l of test sample solutions to 100 l of medium already present in each well . Positive controls dilution (positive control plate)dispense 40 l of stock solution of each positive control (see table 3) in column #2 (figure 4a, wells shaded in red). Dispense 20 l of vehicle in columns #3 and #5 (figure 4a, wells shaded in light blue) and 40 l of vehicle in column #4 (figure 4a, wells shaded in light blue).withdraw 12 l from the wells in column #2, dispense it to the wells in column #3 and mix (figure 4b), continue till a final serial dilution with 3 doses (d1, d2, and d3) and vehicle (v) for each positive control compound is obtained (figure 4c). To generate the positive control plate, prepare a 1:40 dilution of compounds in media (figure 4d).hphc dilution (compound master plate) note: the selected dose ranges of hphcs for hcs are listed in table 2 . Dilute each hphc stock solution (1 m) 1:10 in medium for a concentration of 100 mm . Perform dilutions using medium with 10% vehicle to obtain the selected 5x doses for each hphc . Positive controls dilution (positive control plate)dispense 40 l of stock solution of each positive control (see table 3) in column #2 (figure 4a, wells shaded in red). Dispense 20 l of vehicle in columns #3 and #5 (figure 4a, wells shaded in light blue) and 40 l of vehicle in column #4 (figure 4a, wells shaded in light blue).withdraw 12 l from the wells in column #2, dispense it to the wells in column #3 and mix (figure 4b), continue till a final serial dilution with 3 doses (d1, d2, and d3) and vehicle (v) for each positive control compound is obtained (figure 4c). To generate the positive control plate, prepare a 1:40 dilution of compounds in media (figure 4d). Dispense 40 l of stock solution of each positive control (see table 3) in column #2 (figure 4a, wells shaded in red). Dispense 20 l of vehicle in columns #3 and #5 (figure 4a, wells shaded in light blue) and 40 l of vehicle in column #4 (figure 4a, wells shaded in light blue). Withdraw 12 l from the wells in column #2, dispense it to the wells in column #3 and mix (figure 4b), continue till a final serial dilution with 3 doses (d1, d2, and d3) and vehicle (v) for each positive control compound is obtained (figure 4c). To generate the positive control plate, hphc dilution (compound master plate) note: the selected dose ranges of hphcs for hcs are listed in table 2 . Dilute each hphc stock solution (1 m) 1:10 in medium for a concentration of 100 mm . Perform dilutions using medium with 10% vehicle to obtain the selected 5x doses for each hphc . Dilute each hphc stock solution (1 m) 1:10 in medium for a concentration of 100 mm . Perform dilutions using medium with 10% vehicle to obtain the selected 5x doses for each hphc . Dosing add 25 l of 5x solutions from the compound master plate to the cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 5). Do not remove the existing (100 l) cell culture medium.add 25 l of 5x solution from the positive control plate to the cells in the bottom row maintaining the same dosing order as in the positive control plate (figure 5). Do not remove the existing (100 l) cell culture medium . Incubate the plate at 37 c and 5% co2 for the desired exposure time (4 or 24 hr). Add 25 l of 5x solutions from the compound master plate to the cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 5). Do not remove the existing (100 l) cell culture medium . Add 25 l of 5x solution from the positive control plate to the cells in the bottom row maintaining the same dosing order as in the positive control plate (figure 5). Do not remove the existing (100 l) cell culture medium . Incubate the plate at 37 c and 5% co2 for the desired exposure time (4 or 24 hr). Staining preparation for all assays pre - warm the wash buffer (pbs) solutions at 37 c.prepare the fixation solution (4% formaldehyde solution) adding 10.81 ml of 37% formaldehyde to 89.19 ml of wash buffer and pre - warm it at 37 c.prepare the permeabilization buffer by adding 10 ml of 10x permeabilization buffer to 90 ml of wash buffer and pre - warm it at 37 c.prepare the blocking buffer by adding 10 ml of 10x blocking buffer to 90 ml of wash buffer and pre - warm it at 37 c.pre - warm the cell culture medium at 37 c.remove the hcs plates from the incubator once the 4 and 24 hr exposure times are reached and perform the following specific protocols for each assay . Cytotoxicity assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells)dilute the mitochondria dye in the live cell staining solution according to vendor's instruction . Dilute the membrane permeability dye in the live cell staining solution according to vendor's instruction.add 50 l live cell staining solution to each well of the plate(s) designated " cytotoxicity assay " . Do not remove cell culture medium and incubate for 30 min at 37 c, 5% co2.gently aspirate the medium and staining solution and add 100 l / well of fixation solution to each well, then incubate plate(s) for 20 min at room temperature in the dark.gently aspirate fixation solution and wash once with 100 l / well wash buffer.remove wash buffer and add 100 l / well 1x permeabilization buffer to each well and incubate for 10 min at room temperature in the dark.aspirate permeabilization buffer and wash plate twice with 100 l / well of wash buffer.aspirate wash buffer and add 100 l of 1x blocking buffer to each well and incubate for 15 min at room temperature in the dark.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - cytochrome c antibody (mouse) 1:250 in primary antibody solution.aspirate blocking buffer and add 50 l / well primary antibody solution to each well and incubate for 60 min at room temperature in the dark.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.aspirate primary antibody solution and wash plate three times with 100 l / well of wash buffer using the plate washer.aspirate wash buffer and add 50 l / well of secondary antibody and nuclear solution to each well of the plate(s) and incubate for 60 min at room temperature in the dark.aspirate secondary antibody and nuclear solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Plate(s) is (are) now ready to be evaluated on the hcs reader . Dna damage assay gently aspirate the medium from the plates designated " dna damage".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho h2ax antibody (mouse) 1:2,000 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Stress kinase assay gently aspirate the medium from each of the plates designated " stress kinase".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho cjun antibody (rabbit) 1:200 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - rabbit antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Ros assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the ros dye in live cell staining solution according to vendor instructiondilute the nuclear dye 1:1,000 in live cell staining solution.3.4.5.4) add 50 l live cell staining solution to each well plates designated " ros "; do not remove cell culture media and incubate for 30 min at room temperature in the dark.gently aspirate the medium and staining solution and wash plate three times with 100 l / well medium.aspirate the medium, add 100 l / well fixation solution to each well and incubate plate for 20 min at room temperature in the dark.gently aspirate fixation solution and wash plate three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate the plate on the hcs reader within 1 hr . Gsh content assay prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye (far red) 1:1,000 in live cell nuclear staining solution.3.4.6.3). Add 50 l of live cell nuclear staining solution to each well of the plates designated " gsh content " . Do not remove cell culture media and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell gsh staining solution according to the following formula: volume of hbss (l) v = 50 x w x 1.2 (w number of wells)dilute the gsh dye in live cell gsh staining solution according to vendor's instructions.gently aspirate the medium and the nuclear staining solution and wash plate three times with 100 l / well medium.aspirate medium and add 100 l / well of live cell gsh staining solution to each well of the plate(s).incubate for 10 min at room temperature in the dark . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader within 1 hr . Apoptosis assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the caspase dye 1:300 in live cell staining solution.remove cell culture media, add 50 l live cell staining solution to each well of the plate(s) designated " apoptosis " and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of fix solution (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye 1:1,000 in live cell nuclear staining solution.remove the live cell staining solution, add 100 l / well of live cell nuclear staining solution to each well of the plate(s) and incubate for 30 min at room temperature in the dark.aspirate the fixative / nuclear dye solution and wash plate(s) three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader . Preparation for all assays pre - warm the wash buffer (pbs) solutions at 37 c.prepare the fixation solution (4% formaldehyde solution) adding 10.81 ml of 37% formaldehyde to 89.19 ml of wash buffer and pre - warm it at 37 c.prepare the permeabilization buffer by adding 10 ml of 10x permeabilization buffer to 90 ml of wash buffer and pre - warm it at 37 c.prepare the blocking buffer by adding 10 ml of 10x blocking buffer to 90 ml of wash buffer and pre - warm it at 37 c.pre - warm the cell culture medium at 37 c.remove the hcs plates from the incubator once the 4 and 24 hr exposure times are reached and perform the following specific protocols for each assay . Prepare the fixation solution (4% formaldehyde solution) adding 10.81 ml of 37% formaldehyde to 89.19 ml of wash buffer and pre - warm it at 37 c . Prepare the permeabilization buffer by adding 10 ml of 10x permeabilization buffer to 90 ml of wash buffer and pre - warm it at 37 c . Prepare the blocking buffer by adding 10 ml of 10x blocking buffer to 90 ml of wash buffer and pre - warm it at 37 c . Pre - remove the hcs plates from the incubator once the 4 and 24 hr exposure times are reached and perform the following specific protocols for each assay . Cytotoxicity assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells)dilute the mitochondria dye in the live cell staining solution according to vendor's instruction . Dilute the membrane permeability dye in the live cell staining solution according to vendor's instruction.add 50 l live cell staining solution to each well of the plate(s) designated " cytotoxicity assay " . Do not remove cell culture medium and incubate for 30 min at 37 c, 5% co2.gently aspirate the medium and staining solution and add 100 l / well of fixation solution to each well, then incubate plate(s) for 20 min at room temperature in the dark.gently aspirate fixation solution and wash once with 100 l / well wash buffer.remove wash buffer and add 100 l / well 1x permeabilization buffer to each well and incubate for 10 min at room temperature in the dark.aspirate permeabilization buffer and wash plate twice with 100 l / well of wash buffer.aspirate wash buffer and add 100 l of 1x blocking buffer to each well and incubate for 15 min at room temperature in the dark.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - cytochrome c antibody (mouse) 1:250 in primary antibody solution.aspirate blocking buffer and add 50 l / well primary antibody solution to each well and incubate for 60 min at room temperature in the dark.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.aspirate primary antibody solution and wash plate three times with 100 l / well of wash buffer using the plate washer.aspirate wash buffer and add 50 l / well of secondary antibody and nuclear solution to each well of the plate(s) and incubate for 60 min at room temperature in the dark.aspirate secondary antibody and nuclear solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Plate(s) is (are) now ready to be evaluated on the hcs reader . Prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells) dilute the mitochondria dye in the live cell staining solution according to vendor's instruction . Dilute the membrane permeability dye in the live cell staining solution according to vendor's instruction . Add 50 l live cell staining solution to each well of the plate(s) designated " cytotoxicity assay " . Do not remove cell culture medium and incubate for 30 min at 37 c, 5% co2 . Gently aspirate the medium and staining solution and add 100 l / well of fixation solution to each well, then incubate plate(s) for 20 min at room temperature in the dark . Remove wash buffer and add 100 l / well 1x permeabilization buffer to each well and incubate for 10 min at room temperature in the dark . Aspirate permeabilization buffer and wash plate twice with 100 l / well of wash buffer . Aspirate wash buffer and add 100 l of 1x blocking buffer to each well and incubate for 15 min at room temperature in the dark . Prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - cytochrome c antibody (mouse) 1:250 in primary antibody solution . Aspirate blocking buffer and add 50 l / well primary antibody solution to each well and incubate for 60 min at room temperature in the dark . Prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution . Dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution . Aspirate primary antibody solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Aspirate wash buffer and add 50 l / well of secondary antibody and nuclear solution to each well of the plate(s) and incubate for 60 min at room temperature in the dark . Aspirate secondary antibody and nuclear solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Plate(s) is (are) now ready to be evaluated on the hcs reader . Dna damage assay gently aspirate the medium from the plates designated " dna damage".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho h2ax antibody (mouse) 1:2,000 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - phospho h2ax antibody (mouse) 1:2,000 in primary antibody solution . Prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution . Stress kinase assay gently aspirate the medium from each of the plates designated " stress kinase".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho cjun antibody (rabbit) 1:200 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - rabbit antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - phospho cjun antibody (rabbit) 1:200 in primary antibody solution . Prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - rabbit antibody 1:500 in secondary antibody and nuclear solution . Ros assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the ros dye in live cell staining solution according to vendor instructiondilute the nuclear dye 1:1,000 in live cell staining solution.3.4.5.4) add 50 l live cell staining solution to each well plates designated " ros "; do not remove cell culture media and incubate for 30 min at room temperature in the dark.gently aspirate the medium and staining solution and wash plate three times with 100 l / well medium.aspirate the medium, add 100 l / well fixation solution to each well and incubate plate for 20 min at room temperature in the dark.gently aspirate fixation solution and wash plate three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate the plate on the hcs reader within 1 hr . Prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells). Dilute the ros dye in live cell staining solution according to vendor instruction dilute the nuclear dye 1:1,000 in live cell staining solution.3.4.5.4) add 50 l live cell staining solution to each well plates designated " ros "; do not remove cell culture media and incubate for 30 min at room temperature in the dark . Gently aspirate the medium and staining solution and wash plate three times with 100 l / well medium . Aspirate the medium, add 100 l / well fixation solution to each well and incubate plate for 20 min at room temperature in the dark . Gently aspirate fixation solution and wash plate three times with 100 l / well wash buffer . Gsh content assay prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye (far red) 1:1,000 in live cell nuclear staining solution.3.4.6.3). Add 50 l of live cell nuclear staining solution to each well of the plates designated " gsh content " . Do not remove cell culture media and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell gsh staining solution according to the following formula: volume of hbss (l) v = 50 x w x 1.2 (w number of wells)dilute the gsh dye in live cell gsh staining solution according to vendor's instructions.gently aspirate the medium and the nuclear staining solution and wash plate three times with 100 l / well medium.aspirate medium and add 100 l / well of live cell gsh staining solution to each well of the plate(s).incubate for 10 min at room temperature in the dark . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader within 1 hr . Prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye (far red) 1:1,000 in live cell nuclear staining solution.3.4.6.3). Add 50 l of live cell nuclear staining solution to each well of the plates designated " gsh content " . Do not remove cell culture media and incubate for 30 min at 37 c, 5% co2 . Prepare a sufficient volume (v) of live cell gsh staining solution according to the following formula: volume of hbss (l) v = 50 x w x 1.2 (w number of wells) dilute the gsh dye in live cell gsh staining solution according to vendor's instructions . Gently aspirate the medium and the nuclear staining solution and wash plate three times with 100 l / well medium . Aspirate medium and add 100 l / well of live cell gsh staining solution to each well of the plate(s). Apoptosis assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the caspase dye 1:300 in live cell staining solution.remove cell culture media, add 50 l live cell staining solution to each well of the plate(s) designated " apoptosis " and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of fix solution (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye 1:1,000 in live cell nuclear staining solution.remove the live cell staining solution, add 100 l / well of live cell nuclear staining solution to each well of the plate(s) and incubate for 30 min at room temperature in the dark.aspirate the fixative / nuclear dye solution and wash plate(s) three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader . Prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells). Dilute the caspase dye 1:300 in live cell staining solution . Remove cell culture media, add 50 l live cell staining solution to each well of the plate(s) designated " apoptosis " and incubate for 30 min at 37 c, 5% co2 . Prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of fix solution (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye 1:1,000 in live cell nuclear staining solution . Remove the live cell staining solution, add 100 l / well of live cell nuclear staining solution to each well of the plate(s) and incubate for 30 min at room temperature in the dark . Aspirate the fixative / nuclear dye solution and wash plate(s) three times with 100 l / well wash buffer . Note: raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every end points at a given time point post-dosing.normalize the values using the following equation: where i is the measured raw signal value of a well, vehicle is the median of the measured signal values for the vehicle wells on a plate, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (100), note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that was applied to the sample contained in position / well i and its corresponding normalized signal n(i).plot and fit the (ci, n(i))-values using a 4-parameters hill equation . Where zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Note: ac50 is a measure of potency where low values indicate high potency . Note: raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every end points at a given time point post - dosing . Normalize the values using the following equation: where i is the measured raw signal value of a well, vehicle is the median of the measured signal values for the vehicle wells on a plate, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (100), note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that was applied to the sample contained in position / well i and its corresponding normalized signal n(i). Plot and fit the (ci, n(i))-values using a 4-parameters hill equation . Where zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Pre - warm the cell culture medium (bronchial epithelial cell growth medium - supplemented medium), the hepes, trypsin, and trypsin neutralizing solution (tns) in the water bath at 37 c . Note: the following nhbe cell culture seeding conditions may be used to obtain optimal confluence in uncoated t75 flasks with 20 ml medium: seed 1 x 10 cells for 3-day culture, 0.5 x 10 cells for 4-day culture and 0.25 x 10 cells for 5-day culture . Change medium every 2 days when cells are in culture to refresh nutrients . Seed 1 x 10 cells for 3-day culture, 0.5 x 10 cells for 4-day culture and 0.25 x 10 cells for 5-day culture . Remove the supernatant from the flask(s) and add hepes to wash the cells (e.g., 3 ml for a 75 cm flask). Remove the hepes solution and add trypsin solution (e.g., 3 ml for a 75 cm flask). Rotate the flask to cover the cell monolayer with trypsin solution . Monitor cell detachment under the microscope and if necessary, incubate longer and gently tap the flask to release any remaining attached cells . Add tns to stop the reaction (e.g., 3 ml for a 75 cm flask) and transfer the cell suspension to a 15 ml tube . Discard the supernatant and re - suspend the cell pellet in 10 ml of fresh medium, mixing gently to yield a homogeneous cell suspension . Filter the cell suspension through a 100 m cell strainer to remove aggregates and count the cells . Note: in our laboratory an electric field multi - channel cell counting system was used to precisely and consistently evaluate the viable cells number . Note: an impedance - based measurement system was used to: 1) evaluate compound toxicity, 2) select compounds to be further investigated by hcs and 3) select appropriate doses for hcs . The nhbe cells in the rcta plates are dosed by adding 25 l of test compound dilutions to 100 l medium present in each well . Therefore, all test solutions are prepared at 5 times (5x) the desired final concentration . Seeding nhbe cells program the instrument to define the number and duration of impedance measurements . In this study, data were recorded every 15 min for 48 hr (from 24 hr 2 hr before and 24 hr after dosing the cells with test agents).measure the plate background by pipetting 50 l of pre - warmed medium into each well of a 96-well rtca plate . Note: this step represents a technical requirement for the instrument to calculate the medium electrical resistance which is then used as a baseline reference for cell - based calculation.prepare a cell suspension at a concentration of 144,000 cells / ml (5%) and add 50 l cell suspension (7,200 cells / well) to each well of the rtca plate in which 50 l / well of medium was already added for instrument background measurement.let the cells adhere for 30 min at room temperature before placing them into the rtca cradle (to improve the homogeneous distribution of the cells). Incubate the rtca plates in the rtca cradle in the incubator (37 c and 5% co2) and start recording the data for the next 24 2 hr prior to dosing . Program the instrument to define the number and duration of impedance measurements . In this study, data were recorded every 15 min for 48 hr (from 24 hr 2 hr before and 24 hr after dosing the cells with test agents). Measure the plate background by pipetting 50 l of pre - warmed medium into each well of a 96-well rtca plate . Note: this step represents a technical requirement for the instrument to calculate the medium electrical resistance which is then used as a baseline reference for cell - based calculation . Prepare a cell suspension at a concentration of 144,000 cells / ml (5%) and add 50 l cell suspension (7,200 cells / well) to each well of the rtca plate in which 50 l / well of medium was already added for instrument background measurement . Let the cells adhere for 30 min at room temperature before placing them into the rtca cradle (to improve the homogeneous distribution of the cells). Incubate the rtca plates in the rtca cradle in the incubator (37 c and 5% co2) and start recording the data for the next 24 2 hr prior to dosing . Dilutions of hphcs and positive controls positive control dilution dilute the staurosporine stock solution (10 mm) 1:10 in dmso (see table 3) and add 5 l of the dilution to 195 l of medium to obtain a 5x working solution . Hphc dilution dissolve / dilute each hphc in the vehicle (table 2) to generate a 1 m stock solution . Dilute each hphc stock solution 1:10 in medium to generate a 100 mm solution.generate the " compound master plate " by performing a five - step 1:10 serial dilution using medium + 10% vehicle to obtain the 5x working solutions (figure 2). Note: ultimately, the final doses will be: 0.2 m, 2 m, 20 m, 0.2 mm, 2 mm, 20 mm . Also prepare the dose 0, corresponding to the only vehicle, in this step . Dosing pause the rtca instrument and open the cradle to remove the plate.remove the plate and place it in the rtca plate temperature tool (designed to stabilize the temperature of the rtca plate during experimental procedures outside the rtca station) to avoid cooling the cells, which could impact the impedance measurement.add 25 l 5x solution from the " compound master plate " to cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 2). Do not remove the existing (100 l) cell culture medium.add 25 l 5x positive control solution to the cells in the bottom row (half - right) without removing existing culture medium . Add 25 l medium to the cells in the bottom row (half - left) without removing existing cell culture medium.seal the plate with plate sealer . Note: the use of this sealant film is recommended to avoid potential contamination, including well - to - well cross - contamination . Data analysis rtca and ld50 calculation export raw data as a text (.txt) or excel (.xls) file . Note: the file will contain all the information regarding the plate layout (compounds, doses and well position). Raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every time points at which the recording occurred . The value at position i in the 96 well plate at time t is denoted by cit(i).identify as a normalization reference the latest time point before the dosing for each position i in the 96 well plate (e.g., cell index at 23 hr 50 min 00 sec at position i, cit(i)=23:50:00 = ciref(i)). Note: this information must be annotated when the dosing is performed.divide, on a well base, every time point values by the normalization reference to normalize all the values at the dosing time . The normalized value at position i in the 96 well plate at time t is denoted by ncit(i) and is thus defined by ncit(i) = cit(i) / ciref(i) for all t.calculate the area under the curve (auc) at 24 hr post - dosing for each sample i (position i in the 96 well plate), including positions for positive control and vehicle . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Normalize the values using the following equation: where i is the position in the 96 well plate for which auc was calculated at 24 hr post - dosing, vehicle is the median of the auc values for the vehicle wells on a plate at 24 hr post - dosing, positive control is the median of the auc values for the positive control wells on a plate at 24 hr post - dosing, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (-100%) note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that is applied to the sample contained in position / well i and its corresponding normalized auc at 24 hr post - dosing auc_normalized(i).plot and fit the (ci, auc_normalized(i))-values using a 4-parameters hill equation . Where y = auc_normalized, zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Positive control dilution dilute the staurosporine stock solution (10 mm) 1:10 in dmso (see table 3) and add 5 l of the dilution to 195 l of medium to obtain a 5x working solution . Dilute the staurosporine stock solution (10 mm) 1:10 in dmso (see table 3) and add 5 l of the dilution to 195 l of medium to obtain a 5x working solution . Hphc dilution dissolve / dilute each hphc in the vehicle (table 2) to generate a 1 m stock solution . Dilute each hphc stock solution 1:10 in medium to generate a 100 mm solution.generate the " compound master plate " by performing a five - step 1:10 serial dilution using medium + 10% vehicle to obtain the 5x working solutions (figure 2). Note: ultimately, the final doses will be: 0.2 m, 2 m, 20 m, 0.2 mm, 2 mm, 20 mm . Also prepare the dose 0, corresponding to the only vehicle, in this step . Dissolve / dilute each hphc in the vehicle (table 2) to generate a 1 m stock solution . Generate the " compound master plate " by performing a five - step 1:10 serial dilution using medium + 10% vehicle to obtain the 5x working solutions (figure 2). Note: ultimately, the final doses will be: 0.2 m, 2 m, 20 m, 0.2 mm, 2 mm, 20 mm . Also prepare the dose 0, corresponding to the only vehicle, in this step . Dosing pause the rtca instrument and open the cradle to remove the plate.remove the plate and place it in the rtca plate temperature tool (designed to stabilize the temperature of the rtca plate during experimental procedures outside the rtca station) to avoid cooling the cells, which could impact the impedance measurement.add 25 l 5x solution from the " compound master plate " to cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 2). Do not remove the existing (100 l) cell culture medium.add 25 l 5x positive control solution to the cells in the bottom row (half - right) without removing existing culture medium . Add 25 l medium to the cells in the bottom row (half - left) without removing existing cell culture medium.seal the plate with plate sealer . Note: the use of this sealant film is recommended to avoid potential contamination, including well - to - well cross - contamination . Pause the rtca instrument and open the cradle to remove the plate . Remove the plate and place it in the rtca plate temperature tool (designed to stabilize the temperature of the rtca plate during experimental procedures outside the rtca station) to avoid cooling the cells, which could impact the impedance measurement . Add 25 l 5x solution from the " compound master plate " to cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 2). Add 25 l 5x positive control solution to the cells in the bottom row (half - right) without removing existing culture medium . Add 25 l medium to the cells in the bottom row (half - left) without removing existing cell culture medium . Note: the use of this sealant film is recommended to avoid potential contamination, including well - to - well cross - contamination . Data analysis rtca and ld50 calculation export raw data as a text (.txt) or excel (.xls) file . Note: the file will contain all the information regarding the plate layout (compounds, doses and well position). Raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every time points at which the recording occurred . The value at position i in the 96 well plate at time t is denoted by cit(i).identify as a normalization reference the latest time point before the dosing for each position i in the 96 well plate (e.g., cell index at 23 hr 50 min 00 sec at position i, cit(i)=23:50:00 = ciref(i)). Note: this information must be annotated when the dosing is performed.divide, on a well base, every time point values by the normalization reference to normalize all the values at the dosing time . The normalized value at position i in the 96 well plate at time t is denoted by ncit(i) and is thus defined by ncit(i) = cit(i) / ciref(i) for all t.calculate the area under the curve (auc) at 24 hr post - dosing for each sample i (position i in the 96 well plate), including positions for positive control and vehicle . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Normalize the values using the following equation: where i is the position in the 96 well plate for which auc was calculated at 24 hr post - dosing, vehicle is the median of the auc values for the vehicle wells on a plate at 24 hr post - dosing, positive control is the median of the auc values for the positive control wells on a plate at 24 hr post - dosing, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (-100%) note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that is applied to the sample contained in position / well i and its corresponding normalized auc at 24 hr post - dosing auc_normalized(i).plot and fit the (ci, auc_normalized(i))-values using a 4-parameters hill equation . Where y = auc_normalized, zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Export raw data as a text (.txt) or excel (.xls) file . Note: the file will contain all the information regarding the plate layout (compounds, doses and well position). Raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every time points at which the recording occurred . The value at position i in the 96 well plate at time t is denoted by cit(i). Identify as a normalization reference the latest time point before the dosing for each position i in the 96 well plate (e.g., cell index at 23 hr 50 min 00 sec at position i, cit(i)=23:50:00 = ciref(i)). Divide, on a well base, every time point values by the normalization reference to normalize all the values at the dosing time . The normalized value at position i in the 96 well plate at time t is denoted by ncit(i) and is thus defined by ncit(i) = cit(i) / ciref(i) for all t. calculate the area under the curve (auc) at 24 hr post - dosing for each sample i (position i in the 96 well plate), including positions for positive control and vehicle . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Obtain the auc at position i is obtained by summing the areas of each rectangle, each rectangle being between two timepoints denoted by tk and tl (tk <tl); calculate each rectangle area using xy with x = tl- tk being the distance between two timepoints and y being the mean of the activity of the two timepoints (y = (ncitk(i) + ncitl(i)) / 2). Note: the auc at 24 hr post - dosing for position i is denoted by auc(i). As all the conditions are plated in triplicate wells the median of the three values is used . Normalize the values using the following equation: where i is the position in the 96 well plate for which auc was calculated at 24 hr post - dosing, vehicle is the median of the auc values for the vehicle wells on a plate at 24 hr post - dosing, positive control is the median of the auc values for the positive control wells on a plate at 24 hr post - dosing, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (-100%) note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that is applied to the sample contained in position / well i and its corresponding normalized auc at 24 hr post - dosing auc_normalized(i). Plot and fit the (ci, auc_normalized(i))-values using a 4-parameters hill equation . Where y = auc_normalized, zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Note: a total of nine multi - parametric markers of toxicity, grouped in six different assays, are measured using the hcs platform (table 1). Based on the rtca cell viability analysis (section 2) the dose range of each constituent is defined and a 3r4f reference dose is also included . The reference dose is equivalent to the amount of hphc present in the smoke of one stick from the reference cigarette 3r4f . Seeding nhbe cells prepare a cell suspension at 120,000 cells / ml (5%) and add 100 l cell suspension to each well of a 96-well hcs plate (12,000 cells / well). Prepare enough plates for the assessment of all assays (cytotoxicity, dna damage, stress kinase, ros, gsh content and apoptosis) and timepoints (4 and 24 hr).leave the hcs plates at room temperature for 30 min to allow cells to attach to wells and then incubate at 37 c and 5% co2 for 24 2 hr prior to dosing . Prepare a cell suspension at 120,000 cells / ml (5%) and add 100 l cell suspension to each well of a 96-well hcs plate (12,000 cells / well). Prepare enough plates for the assessment of all assays (cytotoxicity, dna damage, stress kinase, ros, gsh content and apoptosis) and timepoints (4 and 24 hr). Leave the hcs plates at room temperature for 30 min to allow cells to attach to wells and then incubate at 37 c and 5% co2 for 24 2 hr prior to dosing . Dilution of hchps and positive controls note: the nhbe cells in the hcs plates will be dosed by adding 25 l of test sample solutions to 100 l of medium already present in each well . Positive controls dilution (positive control plate)dispense 40 l of stock solution of each positive control (see table 3) in column #2 (figure 4a, wells shaded in red). Dispense 20 l of vehicle in columns #3 and #5 (figure 4a, wells shaded in light blue) and 40 l of vehicle in column #4 (figure 4a, wells shaded in light blue).withdraw 12 l from the wells in column #2, dispense it to the wells in column #3 and mix (figure 4b), continue till a final serial dilution with 3 doses (d1, d2, and d3) and vehicle (v) for each positive control compound is obtained (figure 4c). To generate the positive control plate, prepare a 1:40 dilution of compounds in media (figure 4d).hphc dilution (compound master plate) note: the selected dose ranges of hphcs for hcs are listed in table 2 . Dilute each hphc stock solution (1 m) 1:10 in medium for a concentration of 100 mm . Perform dilutions using medium with 10% vehicle to obtain the selected 5x doses for each hphc . Positive controls dilution (positive control plate)dispense 40 l of stock solution of each positive control (see table 3) in column #2 (figure 4a, wells shaded in red). Dispense 20 l of vehicle in columns #3 and #5 (figure 4a, wells shaded in light blue) and 40 l of vehicle in column #4 (figure 4a, wells shaded in light blue).withdraw 12 l from the wells in column #2, dispense it to the wells in column #3 and mix (figure 4b), continue till a final serial dilution with 3 doses (d1, d2, and d3) and vehicle (v) for each positive control compound is obtained (figure 4c). To generate the positive control plate, dispense 40 l of stock solution of each positive control (see table 3) in column #2 (figure 4a, wells shaded in red). Dispense 20 l of vehicle in columns #3 and #5 (figure 4a, wells shaded in light blue) and 40 l of vehicle in column #4 (figure 4a, wells shaded in light blue). Withdraw 12 l from the wells in column #2, dispense it to the wells in column #3 and mix (figure 4b), continue till a final serial dilution with 3 doses (d1, d2, and d3) and vehicle (v) for each positive control compound is obtained (figure 4c). To generate the positive control plate, hphc dilution (compound master plate) note: the selected dose ranges of hphcs for hcs are listed in table 2 . Dilute each hphc stock solution (1 m) perform dilutions using medium with 10% vehicle to obtain the selected 5x doses for each hphc . Dilute each hphc stock solution (1 m) 1:10 in medium for a concentration of 100 mm . Perform dilutions using medium with 10% vehicle to obtain the selected 5x doses for each hphc . Dosing add 25 l of 5x solutions from the compound master plate to the cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 5). Do not remove the existing (100 l) cell culture medium.add 25 l of 5x solution from the positive control plate to the cells in the bottom row maintaining the same dosing order as in the positive control plate (figure 5). Do not remove the existing (100 l) cell culture medium . Note: each assay has a specific positive control; see table 3 . Incubate the plate at 37 c and 5% co2 for the desired exposure time (4 or 24 hr). Add 25 l of 5x solutions from the compound master plate to the cells in triplicate maintaining the same dosing order as in the compound master plate (highest dose in the top row and vehicle control in the row number 7) (figure 5). Add 25 l of 5x solution from the positive control plate to the cells in the bottom row maintaining the same dosing order as in the positive control plate (figure 5). Incubate the plate at 37 c and 5% co2 for the desired exposure time (4 or 24 hr). Staining preparation for all assays pre - warm the wash buffer (pbs) solutions at 37 c.prepare the fixation solution (4% formaldehyde solution) adding 10.81 ml of 37% formaldehyde to 89.19 ml of wash buffer and pre - warm it at 37 c.prepare the permeabilization buffer by adding 10 ml of 10x permeabilization buffer to 90 ml of wash buffer and pre - warm it at 37 c.prepare the blocking buffer by adding 10 ml of 10x blocking buffer to 90 ml of wash buffer and pre - warm it at 37 c.pre - warm the cell culture medium at 37 c.remove the hcs plates from the incubator once the 4 and 24 hr exposure times are reached and perform the following specific protocols for each assay . Cytotoxicity assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells)dilute the mitochondria dye in the live cell staining solution according to vendor's instruction . Dilute the membrane permeability dye in the live cell staining solution according to vendor's instruction.add 50 l live cell staining solution to each well of the plate(s) designated " cytotoxicity assay " . Do not remove cell culture medium and incubate for 30 min at 37 c, 5% co2.gently aspirate the medium and staining solution and add 100 l / well of fixation solution to each well, then incubate plate(s) for 20 min at room temperature in the dark.gently aspirate fixation solution and wash once with 100 l / well wash buffer.remove wash buffer and add 100 l / well 1x permeabilization buffer to each well and incubate for 10 min at room temperature in the dark.aspirate permeabilization buffer and wash plate twice with 100 l / well of wash buffer.aspirate wash buffer and add 100 l of 1x blocking buffer to each well and incubate for 15 min at room temperature in the dark.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - cytochrome c antibody (mouse) 1:250 in primary antibody solution.aspirate blocking buffer and add 50 l / well primary antibody solution to each well and incubate for 60 min at room temperature in the dark.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.aspirate primary antibody solution and wash plate three times with 100 l / well of wash buffer using the plate washer.aspirate wash buffer and add 50 l / well of secondary antibody and nuclear solution to each well of the plate(s) and incubate for 60 min at room temperature in the dark.aspirate secondary antibody and nuclear solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Plate(s) is (are) now ready to be evaluated on the hcs reader . Dna damage assay gently aspirate the medium from the plates designated " dna damage".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho h2ax antibody (mouse) 1:2,000 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Stress kinase assay gently aspirate the medium from each of the plates designated " stress kinase".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho cjun antibody (rabbit) 1:200 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - rabbit antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Ros assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the ros dye in live cell staining solution according to vendor instructiondilute the nuclear dye 1:1,000 in live cell staining solution.3.4.5.4) add 50 l live cell staining solution to each well plates designated " ros "; do not remove cell culture media and incubate for 30 min at room temperature in the dark.gently aspirate the medium and staining solution and wash plate three times with 100 l / well medium.aspirate the medium, add 100 l / well fixation solution to each well and incubate plate for 20 min at room temperature in the dark.gently aspirate fixation solution and wash plate three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate the plate on the hcs reader within 1 hr . Gsh content assay prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye (far red) 1:1,000 in live cell nuclear staining solution.3.4.6.3). Add 50 l of live cell nuclear staining solution to each well of the plates designated " gsh content " . Do not remove cell culture media and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell gsh staining solution according to the following formula: volume of hbss (l) v = 50 x w x 1.2 (w number of wells)dilute the gsh dye in live cell gsh staining solution according to vendor's instructions.gently aspirate the medium and the nuclear staining solution and wash plate three times with 100 l / well medium.aspirate medium and add 100 l / well of live cell gsh staining solution to each well of the plate(s).incubate for 10 min at room temperature in the dark . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader within 1 hr . Apoptosis assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the caspase dye 1:300 in live cell staining solution.remove cell culture media, add 50 l live cell staining solution to each well of the plate(s) designated " apoptosis " and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of fix solution (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye 1:1,000 in live cell nuclear staining solution.remove the live cell staining solution, add 100 l / well of live cell nuclear staining solution to each well of the plate(s) and incubate for 30 min at room temperature in the dark.aspirate the fixative / nuclear dye solution and wash plate(s) three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader . Preparation for all assays pre - warm the wash buffer (pbs) solutions at 37 c.prepare the fixation solution (4% formaldehyde solution) adding 10.81 ml of 37% formaldehyde to 89.19 ml of wash buffer and pre - warm it at 37 c.prepare the permeabilization buffer by adding 10 ml of 10x permeabilization buffer to 90 ml of wash buffer and pre - warm it at 37 c.prepare the blocking buffer by adding 10 ml of 10x blocking buffer to 90 ml of wash buffer and pre - warm it at 37 c.pre - warm the cell culture medium at 37 c.remove the hcs plates from the incubator once the 4 and 24 hr exposure times are reached and perform the following specific protocols for each assay . Prepare the fixation solution (4% formaldehyde solution) adding 10.81 ml of 37% formaldehyde to 89.19 ml of wash buffer and pre - warm it at 37 c . Prepare the permeabilization buffer by adding 10 ml of 10x permeabilization buffer to 90 ml of wash buffer and pre - warm it at 37 c . Prepare the blocking buffer by adding 10 ml of 10x blocking buffer to 90 ml of wash buffer and pre - warm it at 37 c . Pre - remove the hcs plates from the incubator once the 4 and 24 hr exposure times are reached and perform the following specific protocols for each assay . Cytotoxicity assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells)dilute the mitochondria dye in the live cell staining solution according to vendor's instruction . Dilute the membrane permeability dye in the live cell staining solution according to vendor's instruction.add 50 l live cell staining solution to each well of the plate(s) designated " cytotoxicity assay " . Do not remove cell culture medium and incubate for 30 min at 37 c, 5% co2.gently aspirate the medium and staining solution and add 100 l / well of fixation solution to each well, then incubate plate(s) for 20 min at room temperature in the dark.gently aspirate fixation solution and wash once with 100 l / well wash buffer.remove wash buffer and add 100 l / well 1x permeabilization buffer to each well and incubate for 10 min at room temperature in the dark.aspirate permeabilization buffer and wash plate twice with 100 l / well of wash buffer.aspirate wash buffer and add 100 l of 1x blocking buffer to each well and incubate for 15 min at room temperature in the dark.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - cytochrome c antibody (mouse) 1:250 in primary antibody solution.aspirate blocking buffer and add 50 l / well primary antibody solution to each well and incubate for 60 min at room temperature in the dark.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution . Dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.aspirate primary antibody solution and wash plate three times with 100 l / well of wash buffer using the plate washer.aspirate wash buffer and add 50 l / well of secondary antibody and nuclear solution to each well of the plate(s) and incubate for 60 min at room temperature in the dark.aspirate secondary antibody and nuclear solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Plate(s) is (are) now ready to be evaluated on the hcs reader . Prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells) dilute the mitochondria dye in the live cell staining solution according to vendor's instruction . Dilute the membrane permeability dye in the live cell staining solution according to vendor's instruction . Add 50 l live cell staining solution to each well of the plate(s) designated " cytotoxicity assay " . Do not remove cell culture medium and incubate for 30 min at 37 c, 5% co2 . Gently aspirate the medium and staining solution and add 100 l / well of fixation solution to each well, then incubate plate(s) for 20 min at room temperature in the dark . Remove wash buffer and add 100 l / well 1x permeabilization buffer to each well and incubate for 10 min at room temperature in the dark . Aspirate permeabilization buffer and wash plate twice with 100 l / well of wash buffer . Aspirate wash buffer and add 100 l of 1x blocking buffer to each well and incubate for 15 min at room temperature in the dark . Prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - cytochrome c antibody (mouse) 1:250 in primary antibody solution . Aspirate blocking buffer and add 50 l / well primary antibody solution to each well and incubate for 60 min at room temperature in the dark . Prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells). Dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution . Dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution . Aspirate primary antibody solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Aspirate wash buffer and add 50 l / well of secondary antibody and nuclear solution to each well of the plate(s) and incubate for 60 min at room temperature in the dark . Aspirate secondary antibody and nuclear solution and wash plate three times with 100 l / well of wash buffer using the plate washer . Plate(s) is (are) now ready to be evaluated on the hcs reader . Dna damage assay gently aspirate the medium from the plates designated " dna damage".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho h2ax antibody (mouse) 1:2,000 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - phospho h2ax antibody (mouse) 1:2,000 in primary antibody solution . Prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - mouse antibody 1:500 in secondary antibody and nuclear solution . Stress kinase assay gently aspirate the medium from each of the plates designated " stress kinase".perform the same steps as described in sequence: 3.4.2.4 - 3.4.2.8 . Note that in this instance, only medium is removed during step 3.4.2.4.prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - phospho cjun antibody (rabbit) 1:200 in primary antibody solution.perform the same step as described in 3.4.2.10.prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells)dilute the anti - rabbit antibody 1:500 in secondary antibody and nuclear solution.dilute the nuclear dye 1:1,000 in secondary antibody and nuclear solution.perform the same steps as described in sequence: 3.4.2.12-3.4.2.14 . Prepare a sufficient volume (v) of primary antibody solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - phospho cjun antibody (rabbit) 1:200 in primary antibody solution . Prepare a sufficient volume (v) of secondary antibody and nuclear solution according to the following formula: volume of blocking buffer (l) v = 50 x w x 1.2 (w number of wells) dilute the anti - rabbit antibody 1:500 in secondary antibody and nuclear solution . Ros assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the ros dye in live cell staining solution according to vendor instructiondilute the nuclear dye 1:1,000 in live cell staining solution.3.4.5.4) add 50 l live cell staining solution to each well plates designated " ros "; do not remove cell culture media and incubate for 30 min at room temperature in the dark.gently aspirate the medium and staining solution and wash plate three times with 100 l / well medium.aspirate the medium, add 100 l / well fixation solution to each well and incubate plate for 20 min at room temperature in the dark.gently aspirate fixation solution and wash plate three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate the plate on the hcs reader within 1 hr . Prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells). Dilute the ros dye in live cell staining solution according to vendor instruction dilute the nuclear dye 1:1,000 in live cell staining solution.3.4.5.4) add 50 l live cell staining solution to each well plates designated " ros "; do not remove cell culture media and incubate for 30 min at room temperature in the dark . Gently aspirate the medium and staining solution and wash plate three times with 100 l / well medium . Aspirate the medium, add 100 l / well fixation solution to each well and incubate plate for 20 min at room temperature in the dark . Gently aspirate fixation solution and wash plate three times with 100 l / well wash buffer . Gsh content assay prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye (far red) 1:1,000 in live cell nuclear staining solution.3.4.6.3). Add 50 l of live cell nuclear staining solution to each well of the plates designated " gsh content " . Do not remove cell culture media and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell gsh staining solution according to the following formula: volume of hbss (l) v = 50 x w x 1.2 (w number of wells)dilute the gsh dye in live cell gsh staining solution according to vendor's instructions.gently aspirate the medium and the nuclear staining solution and wash plate three times with 100 l / well medium.aspirate medium and add 100 l / well of live cell gsh staining solution to each well of the plate(s).incubate for 10 min at room temperature in the dark . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader within 1 hr . Prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye (far red) 1:1,000 in live cell nuclear staining solution.3.4.6.3). Add 50 l of live cell nuclear staining solution to each well of the plates designated " gsh content " . Do not remove cell culture media and incubate for 30 min at 37 c, 5% co2 . Prepare a sufficient volume (v) of live cell gsh staining solution according to the following formula: volume of hbss (l) v = 50 x w x 1.2 (w number of wells) dilute the gsh dye in live cell gsh staining solution according to vendor's instructions . Gently aspirate the medium and the nuclear staining solution and wash plate three times with 100 l / well medium . Aspirate medium and add 100 l / well of live cell gsh staining solution to each well of the plate(s). Apoptosis assay prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells).dilute the caspase dye 1:300 in live cell staining solution.remove cell culture media, add 50 l live cell staining solution to each well of the plate(s) designated " apoptosis " and incubate for 30 min at 37 c, 5% co2.prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of fix solution (l) v = 50 x w x 1.2 (w number of wells).dilute the nuclear dye 1:1,000 in live cell nuclear staining solution.remove the live cell staining solution, add 100 l / well of live cell nuclear staining solution to each well of the plate(s) and incubate for 30 min at room temperature in the dark.aspirate the fixative / nuclear dye solution and wash plate(s) three times with 100 l / well wash buffer.add 100 l / well wash buffer . Plate(s) is (are) now ready.evaluate plate(s) on the hcs reader . Prepare a sufficient volume (v) of live cell staining solution according to the following formula: volume of medium (l) v = 50 x w x 1.2 (w number of wells). Dilute the caspase dye 1:300 in live cell staining solution . Remove cell culture media, add 50 l live cell staining solution to each well of the plate(s) designated " apoptosis " and incubate for 30 min at 37 c, 5% co2 . Prepare a sufficient volume (v) of live cell nuclear staining solution according to the following formula: volume of fix solution (l) v = 50 x w x 1.2 (w number of wells). Dilute the nuclear dye 1:1,000 in live cell nuclear staining solution . Remove the live cell staining solution, add 100 l / well of live cell nuclear staining solution to each well of the plate(s) and incubate for 30 min at room temperature in the dark . Aspirate the fixative / nuclear dye solution and wash plate(s) three times with 100 l / well wash buffer . Note: raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every end points at a given time point post-dosing.normalize the values using the following equation: where i is the measured raw signal value of a well, vehicle is the median of the measured signal values for the vehicle wells on a plate, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (100), note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that was applied to the sample contained in position / well i and its corresponding normalized signal n(i).plot and fit the (ci, n(i))-values using a 4-parameters hill equation . Where zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Note: ac50 is a measure of potency where low values indicate high potency . Note: raw cell index values are organized in a 96 well format (mirroring plate well distribution) and are provided for every end points at a given time point post - dosing . Normalize the values using the following equation: where i is the measured raw signal value of a well, vehicle is the median of the measured signal values for the vehicle wells on a plate, cr is the desired median normalized value for the vehicle (0%), and sc is the desired median normalized value for the positive controls (100), note: at the end of this step, a data set is obtained that contains, for each position i in the 96 well plate, a concentration ci (in logarithmic units) that was applied to the sample contained in position / well i and its corresponding normalized signal n(i). Plot and fit the (ci, n(i))-values using a 4-parameters hill equation . Where zero activity s0 = activity level at zero concentration of test compound, infinite activity sinf = activity level at infinite concentration, ac50 = concentration at which activity reaches 50% of maximum level, hill coefficient n = measure of the slope at ac50, and c = concentration in logarithmic units corresponding to the values on the x - axis of the dose - response curve plot . Because the hcs endpoints will not be informative when no toxic effect is detected, those compounds not showing decreased cell viability up to the highest concentration in the rcta are not tested by hcs (figure 3b, c, d, g, k, l, m, p). Compounds showing decreased cell viability at only the highest concentration (figure 3e, o) are also deselected for hcs . Finally, only the constituents with a computable ld50 (<20 mm) are selected for further hcs analysis (figure 3a, f, h, j, n). Hphcs meeting the above criteria are: 1-aminonaphtalene, arsenic (v), chromium (vi), crotonaldehyde and phenol . As a quality check (qc) no dose - response curves are plotted as only three doses are tested and not all the three doses are considered at every time - point . Positive control doses are selected (based on previous experiments, data not shown) so that appropriate responses are observed for each endpoint at both 4 hr and 24 hr . In particular doses 1 and 2 are used to evaluate the effect at 4 hr while doses 2 and 3 are used to evaluate the effect at 24 hr . Note that for all the endpoints, except mitochondrial membrane potential and gsh content, an increase of the signal intensity is expected . All compounds, except for phenol, induced a necrotic phenotype, based on increased cell membrane permeability (figure 7a, f, h, l). 1-aminonaphtalene, chromium (vi), crotonaldehyde and phenol were identified as being genotoxic based on increased phosphorylation of the histone h2ax (figure 7e, j, n, p). Phenol and 1-aminonaphtalene were found to induce severe mitochondrial dysfunction (figure 7b, o) which, with 1-aminonaphtalene, led to an increased cytochrome c release (figure 7c). Oxidative stress induction (ros or gsh) was also detected upon treatment with 1-aminonaphtalene, crotonaldehyde and phenol (figure 7d, m, q). Finally, arsenic induces cell stress as demonstrated by the increased phosphorylation of the transcription factor cjun (figure 7 g). First, a dose - range finding was performed using the rtca platform to select appropriate doses for subsequent hcs to characterize the compound - specific toxicity profiles . 24 hr after seeding, cells were dosed and impedance values continuously monitored over the following 24 hr, whereas hcs endpoints were investigated 4 and 24 hr after dosing . Compound master plate is first generated by performing a five - step 1:10 serial dilution . Each compound, including the vehicle control (dose 0) is then added in triplicate to the exposure plate together with medium and staurosporine as controls . Note that the doses sequence is maintained upon transfer, highest doses are in row number 1 while vehicle controls are in row number 7 . Representative rtca cell viability results.a) 1-aminonaphtalene, b) 2-nitropropane, c) acetamide, d) acetone, e) acrylamide, f) arsenic (v), g) benzene, h) chromium (vi), j) crotonaldehyde, k) methyl ethyl ketone, l) nickel (ii), m) nitrobenzene, n) phenol, o) quinoline, p) toluene . At 24 hr post - dosing, area under the curve (auc) was calculated for each dose (including positive control and vehicle) and normalized in a range from 0 to -100% activity (y - axis), where 0 reflects the activity of the vehicle and -100 of the positive control . Values were then plotted and fitted using a four - parameter hill equation and, when possible, ld50 was calculated . Dilution scheme for positive control compounds for hcs assays.a) addition of positive controls and vehicle to the serial dilution plate . D - e) dilution of the 200x positive controls doses in medium (1:40) to generate the positive control plate containing the 5x doses . Note that each doses is diluted in triplicates to reflect the final layout in the exposure plate). Each compound, including the vehicle control (dose 0) is then added in triplicate to the exposure plate together with the positive controls . Note that the doses order is maintained upon transfer, highest doses are in row number 1 while vehicle controls are in row number 7 . Representative fluorescent photos of antibody- or dye - stained cells.a)nuclear parameters - nuclear dye: a permeable dye which binds to dna in live or fixed cells . B)necrosis - cell membrane permeability dye: dye - based detection of cell membrane integrity . The membrane becomes permeable and the dye enters the cell and binds to dna producing a strong fluorescent signal . C)apoptosis - cytochrome c: antibody - based detection of cytochrome c release, a well - known hallmark of early apoptosis . Upon induction of apoptosis, cytochrome c is released from the mitochondria and diffuses into the nucleus . D)dna damage - ph2ax: antibody - based detection of phosphorylation of histone h2ax, a well - known hallmark of double strand dna breaks . E)stress kinase - cjun: antibody - based detection of phosphorylation at ser-73 of cjun, a well - known hallmark of cellular stress . Dihydroethidium itself fluoresces blue in the cytoplasm while the oxidized form ethidium fluoresces red upon dna intercalation . H) apoptosis - caspase 3/7 activation: dye - based detection of caspase 3/7 activity . Reagent is non - fluorescent with a four amino acid peptide that inhibits dna binding . Upon caspase-3/7 activation, the peptide is cleaved enabling the dye to bind to dna and produce a bright, fluorogenic response . 1-aminonaphtalene (a - e), arsenic (v) (f and g), chromium (vi) (h - k), crotonaldehyde (l - n) and phenol (o - q). 4 hr (blue line) and 24 hr (orange line) signals were calculated for each doses and normalized to the vehicle activity (0%). Table 2 . List of tested hphc compounds with relative ld50 at 24 hr of treatment . Compounds selected for hcs analysis are highlighted in orange and doses tested are also given . The 3r4f dose is equivalent to the amount of constituent present in the smoke of one stick from the reference cigarette 3r4f . The needs for alternatives to animal experimentation and for new high throughput testing approaches have been widely discussed over the past years . This has led scientists and regulatory authorities to investigate alternative methods for standard toxicity testing, utilizing cellular assays that closely mimic the physiology of target tissues . In this study, we have demonstrated the applicability of combining a real - time cell analyzer (rtca) with a high content screening (hcs) platform to assess the impact of exposure to single cs constituents on human lung epithelial cells . This setup could be analogously applied to evaluate cytotoxicity induced by various other airborne pollutants, airborne particles, and nanoparticles . Furthermore, the obtained results can be matched with those from whole - genome transcriptomics and computational methods based on causal biological networks . As previously reported, this approach allowed us to corroborate data on molecular pathway perturbation upon cs exposure with hcs endpoints, addressing these pathway perturbations also phenotypically . As a flowchart assay, real - time cell analysis provides cell viability - related information in a dose- and time - dependent resolution, which allows better decision making which dose and exposure time point may be favorable for downstream analysis . The principle of the analyzer relies on changes in electrical impedance generated by the cells as they attach and spread on a culture well surface covered with a gold microelectrode . The impedance is converted into a dimensionless parameter named cell - index, which can be used to monitor cell adhesion, spreading, morphology and ultimately cell viability . Though this technique does not provide information on cytotoxic mechanisms, its sensitivity enables detection of morphological cellular changes even at very low doses at which the hcs is not informative (data not shown). Based on previous experiments, we have noted that rtca methodology is able to detect morphological changes at lower doses compared to the hcs endpoints . Following initial screening with the real - time cell analyzer, a hcs platform was used to gain more in - depth information on the kind of cytotoxicity elicited by each hphc . The hcs assay panel allowed to profile hphcs towards their potential impact on cellular compartments / organelles as well as to identify those eliciting genotoxicity or oxidative stress . In general, all the compounds, except phenol, were found to induce necrosis at the highest tested doses . Consistent with a potential role in cancer development 1-aminonaphtalene induced phosphorylation of h2ax as a marker for genotoxicity, however the hcs panel also uncovered activity of this hphc in the mitochondrial toxicity readout (mass increased and cytochrome c release) and oxidative stress (gsh depletion). Similarly, as previously described, phenol was identified to induce mitochondrial dysfunction, and cause dna damage as well as gsh depletion . Chromium (vi), one of the compounds classified as group i carcinogens, and crotonaldehyde were also both identified as genotoxic, in particular chromium (vi) also induced apoptosis (caspase cascade activation) and crotonaldehyde caused increased ros generation . Finally arsenic (v), was found to induce cjun phosphorylation which is a marker of stress kinase pathway activation . In this study, we utilized nhbe cells as a model for lung epithelial cells in vitro . Using these cells in a hcs setting is unprecedented and enabled the investigation of a broader range of endpoints, including genotoxicity and oxidative stress markers . Both live cell and fixed cell staining approaches were described within our protocols, demonstrating the flexibility of the overall technique . In fact, the very same protocols can be applied to a broader range of targets, which can be addressed by the use of any fluorescent dye or antibody . For the successful execution of the live staining protocols, it is important to respect the incubation time, as some of the dyes have a limited half - life and the fluorescence signal may decrease before the image acquisition is completed . It is also important to consider that if a different cell type is used, all the staining conditions should be re - evaluated, as the optimal dye concentration and the incubation time may be different . In the current paper we have described a scenario where only five compounds where screened with the hcs methodology . Considering the previously described plate layout, they were dosed over 2 different plate sets for a total of 24 plates (6 assays and 2 time - points).the number of plates could also be increased, thereby allowing for the simultaneous screening of more compounds or the investigation of more endpoints . Before doing so, however, one should take into consideration that certain endpoints (gsh and ros) require immediate acquisition, and as a consequence, the dosing of the plates should be performed in a staggered fashion to permit the acquisition of the previous plate . On the other hand, using a fixed cell staining protocol represents an advantage as the plates can be stacked, interrupting the protocol at any step after the fixation, for completion of the staining procedure at a later stage . This approach, for example, would provide the operator with the time to complete all live cell staining plates without compromising the data quality . To further optimize the workflow by decreasing the number of plates, it would also be possible to multiplex more endpoints together . For example in this context dna damage and stress kinase could be investigated together simply using two secondary antibody with fluorochromes emitting in different channels . Continuous development of the hcs platform, including fully automated cell seeding, compound dilution, dosing and staining, as well as the addition of new endpoints will further expand the capability of the hcs platform as a powerful profiling tool for hphcs on epithelial and other cell types.
Regular exercise offers protection against and may be useful as a treatment for a wide variety of chronic diseases associated with low - grade inflammation [13]. Indeed, exercise triggers important adaptations in the inflammatory system, which vary depending on the type and duration of the exercise intervention . Although the inflammatory response to acute exercise may not fully resemble disease associated inflammation, it represents a well - established and useful model to investigate the mechanisms controlling inflammation . In particular, studies employing eccentric exercise have highlighted the impact of this type of exercise on the inflammatory response . Thus, an acute bout of eccentric exercise induces a marked proinflammatory response, while an eccentric training program attenuates the activation of different signaling pathways involved in inflammatory processes [68]. Recently, the regulation of toll - like receptors (tlrs) as key factors involved in the inflammatory responses to exercise has received great attention [9, 10]. Tlrs are type - i transmembrane glycoproteins expressed in different cells of the innate immune system and in other cell types, such as fibroblasts or epithelial cells . Tlrs mediate the recognition of pathogen - associated molecular patterns and play an important role in the immune and inflammatory responses . Among all the tlrs described, tlr4 has been shown to highly respond to different exercise protocols [6, 1214]. In conjunction with cd14, tlr4 orchestrates several processes in the inflammatory cascade and can activate different signaling pathways that increase, or inhibit, the activity of several transcription factors . Three major transcription factors are controlled, at least to some extent, by tlr4 signaling pathways: nuclear transcription factor b (nf-b), activator protein-1 (ap-1), and interferon regulatory transcription factor 3 (irf3). A recent study showed that eccentric exercise plays an important role in tlr4-induced nf-b activation through both the myeloid differentiation factor 88- (myd88-) dependent and independent pathways in peripheral blood mononuclear cells (pbmc) of young men . However, data explaining possible exercise - induced tlr4 regulation of ap-1 and irf3 are scarce . Most of the tlr4 signaling data have been collected with elderly populations [10, 1618], and although few reports include young men [6, 19], tlr4 response to exercise is practically unknown in young women . This may seem surprising given that women are more susceptible to autoimmune and inflammatory diseases, probably due to their higher basal inflammatory status [20, 21]. In fact, the sex difference in subcutaneous adiposity and a differential regulation of cytokine production are probably important factors explaining the sex differences in proinflammatory markers concentrations [22, 23]. Given the great benefits of exercise as a potential tool to protect against the development of chronic inflammatory diseases later in life, research assessing exercise - induced tlr4 activity in young women is warranted . First, we aimed to describe the regulation of nf-b, irf3, and indirectly ap-1 through tlr4 signaling pathway activation in pbmc in response to an acute bout of eccentric exercise in young female subjects . Considering that tlr4 signaling was upregulated in men after a similar bout of exercise, we hypothesized that the acute eccentric bout would induce an upregulation of the tlr4-related pathways . In addition, to further understand the effects of eccentric training on tlr4 regulation, we evaluated the influence of this training paradigm on the activity pattern of different tlr4-initiated pathways . It was hypothesized that the activation of the different tlr4 signaling pathways, for example, nf-b, ap-1, and irf3, in response to an acute eccentric bout would be reduced after the eccentric training period . All the activities involved in this study, including familiarization sessions, were performed during a 12-week period . Initially, two groups of young women completed maximal strength tests, followed by an acute eccentric bout some days later . After 2 weeks, one of the groups (training group, tg) followed a 6-week eccentric training program, while the rest of the participants (control group, cg) followed their daily routines . One week after the last training session, all the subjects repeated the strength tests and the acute eccentric bout . Blood samples were obtained before and immediately after and 2 h after each acute eccentric bout . Peripheral blood mononuclear cells (pbmc) all subjects were healthy sport science undergraduate students involved in recreational physical activities 35 h per week (team and ball sports). None of them reported previous or present resistance exercise training experience or muscle joint or bone injuries . Subjects reporting inflammatory or autoimmune conditions or taking any medication known to affect their inflammatory, hormonal, or metabolic response were excluded from the study . None of the participants were smokers . To minimize the effects of different sex hormone concentrations, for example, estrogens may modulate innate immunity both at rest and during exercise [20, 23], all the volunteers from the present investigation carried out the first and the second (1 week) acute eccentric bouts during the follicular phase of the menstrual cycle (self - reported). This phase has been described as an appropriate moment to evaluate the effects of exercise in women [23, 25]. Participants were randomly assigned to either a training group (tg; n = 12) or to a control group (cg; n = 8). Age, height, body mass, body mass index, and fat% (analyzed by skinfold measurements using yuhasz equation) were 22.5 0.3 yrs, 163.8 1.4 cm, 60 2.1 kg, 22.4 3.2 kg / m, and 18.5 2.8% fat, respectively, in women from tg and 22.5 2.3 yrs, 160.7 1.8 cm, 59.7 3.1 kg, 23.0 2.5 kg / m, and 17.9 3.1% fat, respectively, in women form cg . The purposes and possible risks associated with participation in the study were explained to the subjects before written consent for participation was obtained . The study followed the principles of the declaration of helsinki, and all procedures were approved by the local ethics committee . Maximal strength tests took place after two familiarization sessions, where appropriate squat exercise technique was explained . Three to five days before the first and the second acute eccentric bouts all participants performed a maximal voluntary isometric contraction (mvic) and a one repetition maximum (1rm) tests using a multipower device (e.g., guided barbell squat exercise, salter, barcelona, spain). After a standardized warmup, subjects carried out the mvic test in a squat position, with 110 knee flexion . Each participant completed two mvic tests lasting 5 seconds, with 1 min of rest in between . Force was registered using a strain gauge (globus ergometer, codogn, italy). If the values obtained after the two trials differed more than 5%, a third test was performed . Briefly, participants had to lift an estimated load from 90 knee flexion to full extension (180). The load was increased 10 kg if the participant succeeded or decreased 5 kg if they failed . The protocol followed during the acute eccentric - damaging bouts was similar to fernndez - gonzalo et al . And garca - lpez et al . The eccentric insult corresponded to the negative phase of the squat exercise performed using a guided barbell multipower device . The bout comprised 12 sets of 10 repetitions with a load equivalent to 60% of the mvic . A 3-min rest period was allowed between sets . Once the subject completed the eccentric action (lower the load from 180, full extension, to 90 knee flexion), research assistants raised the load up using a pulley system . Subjects lowered the load with the most comfortable velocity for them, although they were requested to control the descent of the barbell during the full range of motion, allowing the subjects to stop the movement at ~90 knee flexion . An encoder system (globus real power) was used to register distance, time, and velocity of the vertical displacements of the barbell . The first acute eccentric bout was performed ~2 weeks before the first training session, whereas the second bout was carried out ~1 week after the last training session . All participants completed both acute eccentric bouts . Over 6 weeks, tg subjects completed 18 training sessions (3 sessions per week) with at least 48 h between sessions . The training program was initiated ~2 weeks after the first acute eccentric bout and it was completed ~1 week before the second acute eccentric bout . The eccentric action (full extension to 90 knee flexion) and the multipower device described for the acute eccentric bouts were used during the training sessions . The velocity of each eccentric action was monitored to offer real - time feedback to the subjects, allowing them to perform the movement with a similar velocity to the initial acute bout . The load and volume of the training was progressively increased in a weekly manner: weeks 1 and 2: 3 10 and 5 10 at 40% of mvic, respectively; weeks 3 and 4: 3 10 and 5 10 at 45% of mvic, respectively; weeks 5 and 6: 3 10 and 5 10 at 50% of mvic, respectively . Blood samples (30 ml) were obtained from the brachiocephalic vein before, immediately after, and 2 h after each acute eccentric bout using vacutainer system (bd, franklin lakes, nj) with edta . Basal blood collection was performed under conditions of fasting and rest (8:00 a.m). All participants remained in the exercise physiology laboratory at the university of len until finishing acute testing and sampling . During this time, the subjects ingested only water . Relative quantity of pbmc subpopulations was assessed using an automatic analyzer in 5 ml of total blood (technicon h1, bayer, tarrytwon, ny). Density gradient centrifugation on ficoll separating solution (biochrom ag, berlin, germany) was employed to separate pbmc from total blood . Total rna was isolated from pbmc using a ribopuretm - blood kit (ambion, paisley, uk) and quantified by spectrophotometry (nanodrop 1000, thermo scientific, waltham, ma, usa). Dnase i (rnase - free) (ambion) was used to removed residual genomic dna . First - standard cdna was synthesized using high - capacity cdna archive kit (applied biosystems, paisley, uk) and then it was amplified using taqman universal pcr master mix (applied biosystems) on a steponeplustm real - time pcr systems (applied biosystems). Taqman primers and probes for cd14 (genbank m86511.1 and hs00169122_g1), tlr4 (genbank u88880.1 and hs00370853_m1), traf6 (genbank bc031052.1 and hs00377558_m1), and 18srrna as housekeeping gene (genbank x03205.1 and hs99999901_s1) were derived from the commercially available taqman assays - on - demand gene (applied biosystems). Relative changes in gene expression levels were determined using the 2-ct method as described previously . The cycle number at which the transcripts were detectable (ct) was normalized to the cycle number of gapdh detection, referred to as ct . Lysate proteins were fractionated by sds - page and western blotting was performed using the corresponding primary antibodies . Antibodies against tlr4 (96 kda), myd88 (33 kda), p65 (65 kda), perk (4244 kda), tbk1 (80 kda), irf3 (50 kda), protein c - reactive (pcr) (30 kda), and tnf (27 kda) were purchased from santa cruz biotechnology (santa cruz, ca); antibodies against cd14 (40 kda), traf6 (55 kda), trif (66 kda), and ikki / ikk (80 kda) were purchased from abcam (cambridge, uk); and antibodies against pikk (8587 kda), pib (40 kda), p38 (43 kda), pp38 (43 kda), and pirf3 (4555 kda) were purchased from cell signaling technology (beverly, ma). Equal loading of protein was demonstrated by probing the membranes with a rabbit antilamin - b polyclonal antibody (santa cruz biotechnology) (67 kda) or rabbit anti--actin polyclonal antibody (sigma - aldrich) (42 kda). The density of the specific bands was quantified with an imaging densitometer (image j, national institute of health, bethesda, md). Data are expressed as means of percentages from resting values standard error of means (sem). Shapiro - wilk test was used to verify normal data distribution . To compare differences within each group, data were analyzed using an analysis of variance (anova) with repeated measures for training (pre- and posttraining) and time (baseline, immediately, and 2 h after acute bout). To compare results between tg and cg after the first and the second acute eccentric bout, a two - way anova with repeated measurements for group (tg and cg) and time (baseline, immediately, and 2 h after acute bouts) was used . All analyses were performed with spss version 17.0 statistical software (chicago, il). The 6-week eccentric training program took place during the 9-week interval between the two acute eccentric bouts . At baseline, there were no differences in 1rm or mvic between groups (p> 0.05). One repetition maximum (126 4 versus 140 5 kg) and mvic (140 4 versus 159 3 kg) increased in tg after the training program, but not in cg (137 7 versus 139 7 kg and 149 8 versus 142 7 kg, for 1rm and mvic, resp). No differences between exercise bouts or groups were found in the distribution of pbmc subpopulations (table 1). Muscle soreness (mm in vas) before and 48 hours after the first acute eccentric bout was 2.9 5.3 and 43.6 16.9, respectively, for tg and 2.5 4.3 and 49.3 16.3, respectively, for cg . Compared with the first, soreness was lower in both groups (p <0.05) 48 hours after the second acute eccentric bout (13.0 5.7 for tg; 31.7 9.1 for cg), and the decrease was greater for the tg (p <0.001). Cd14 and tlr4 mrna levels increased after both acute eccentric bouts for tg and cg (p <0.04), with no differences between bouts or groups (figures 1(a) and 1(b)). The first acute bout of eccentric exercise induced an increase of cd14 and tlr4 protein content in cg and tg that was significant immediately after exercise (p <0.05) and after 2 h (p <0.05). Similar results were observed after the second bout in cg group . However, after the training program no significant change was detected following the eccentric bout in tg (figures 1(c) and 1(d)). Myd88 (figure 2(a)) increased after the first acute bout in tg and cg (p <0.02). However, this protein was downregulated after the second bout in tg when compared with the first eccentric bout and with cg (p <0.03). Similarly, trif (figure 2(b)) also increased significantly (p <0.03) in response to the first acute eccentric bout in both cg and tg, but only in cg after the second bout . Trif protein expression was significantly reduced after the second bout in tg (p <0.01) when compared with the first bout or with cg . Traf6 mrna levels were also analyzed . In the same line with cd14 and tlr4, traf6 mrna levels (figure 3(a)) increased after both acute eccentric bouts for tg and cg (p <0.05). The first acute eccentric bout triggered a significant (p <0.04 immediately after and p <0.03 2 h after the first acute eccentric bout) increase in traf6 protein concentration (figure 3(b)) in tg and cg . After the second acute bout, cg still showed such upregulation, while tg values did not differ from basal . Phospho - ib protein levels (figure 3(c)) increased significantly (p <0.02) in response to the first acute eccentric bout in both cg and tg . Similar results were observed after the second bout in cg group, but a reduction in phospho - ib was found after the second bout in tg (p <0.01) when compared with the first bout or with cg . Figure 3(d) shows p65 protein content in nuclear extracts, which increased progressively reaching maximal levels 2 h after the first acute bout (p <0.01). Posttraining values were lower in tg group, but they remained elevated in cg after the second acute bout . A marked increase of cytosolic phospho - erk-1/2 protein concentration was evident immediately after the first acute eccentric exercise and was maintained 2 h after (p <0.01 and p <0.01, resp .) In cg and tg group (figure 3(e)). However, 6 weeks of eccentric training reduced the phosphorylation of erk-1/2 to basal values after the acute bout in tg . Tnf and crp protein levels were measured as an estimation of the proinflammatory status (figures 4(a) and 4(b), resp). Tnf increased after the first acute eccentric bout in both tg and cg (p <0.04), but only in cg after the second bout (p <0.04). Furthermore, tg showed lower tnf protein expression after the second acute eccentric bout when compared with the first bout immediately after and 2 h after, and with cg 2 h after the bout (p <0.05). The first acute eccentric bout also triggered a significant increase of crp (p <0.04) in tg and cg . After the second acute bout, cg still showed upregulation of crp (p <0.04), while tg values were markedly reduced after the second bout (p <0.05). The inducible ikk (figure 5(a)) increased immediately after the first acute eccentric bout and was maintained 2 h after in both groups (p <0.04). After the second bout this upregulation persisted in cg but was blunted in tg (p <0.03). No changes were observed in the phosphorylation state of irf3 in response to acute exercise either before or after 6 weeks of training (figure 5(b)). The current study investigated the effects of eccentric exercise on the tlr4-mediated inflammatory response in healthy young women . The results indicate that 6 weeks of submaximal eccentric training attenuate several intermediates related to the nf-b and ap-1 activation and, consequently, the proinflammatory response detected after an acute bout of eccentric exercise . This response was associated with a downregulation of the tlr4 signaling through both myd88-dependent and independent pathways . Our data further suggest that perhaps other tlr4 signaling pathways, such as irfs, could be implicated in the beneficial adaptations induced by training . Previous studies have shown that tlr4 may play a critical role as a link between inflammatory cytokine production and a physically active lifestyle [6, 9, 17]. Thus, resistance - trained older women had significantly lower inflammation and tlr4 mrna levels than sedentary counterparts . Moreover, physical activity status, but not age, influenced tlr4 cell - surface expression . Our results confirm that a single bout of eccentric exercise increases tlr4 and cd14 mrna and protein contents in young women, as it had previously been reported in men . Interestingly, only the protein content overexpression of the receptor and coreceptor in pbmc was lower following the eccentric training program . Therefore, these data provide further support for a training - induced downregulation of tlr4 and its coreceptor cd14 at protein level . Confirming previous results in young men, there was a discrepancy between cd14 and tlr4 mrna levels and protein concentration, which may indicate posttranscriptional adaptations of cd14 and tlr4 in response to eccentric training . Different stimuli induce tlr4 activation via recruitment of different adaptor / signaling molecules to the cytoplasmic domain of the receptor, thus resulting in different cellular responses . In addition, the cell surface or endosomal localization of tlr4 drives recruitment of myd88 or toll / interleukin-1 receptor (tir) domain containing adapter inducing interferon- (trif), respectively . In the present study, an acute eccentric bout activated both the myd88-dependent pathway and the trif - dependent pathway, which mediate the early- and late - phase activation of nf-b, respectively . This activation was blunted in young women after 6 weeks of training, which resembles previous reports in both human and animal models [6, 31]. Early- and late - phase activation of nf-b through tlr4 signaling is mediated by tnf receptor - associated factor 6 (traf6). Traf6 recruits the protein kinase complex tak1 (transforming growth factor beta - activated kinase-1) and tabs (tak1 binding proteins) [33, 34], which then activate two distinct pathways involving the ib kinases (ikk) complex (nf-b activation) and the mitogen - activated protein kinase (mapk) (erk, jnk, p38) pathway, for example, ap-1 activation . The results from the present study indicate that an acute bout of eccentric exercise induced an increase of traf6 protein concentration, which was associated with ikk and mapk signaling pathway activation . This resembles previous observations where eccentric contractions also induced a significant increase in nf-b activation [7, 8] and mapkerk1/2 phosphorylation . Remarkably, the eccentric training reduced the protein concentration of traf6, phospho - ib, p65, and mapkerk1/2 in tg after the second acute eccentric bout compared with the first bout and with cg . The proinflammatory cytokine tnf-, which is regulated by transcription factors such as nf-b and ap-1, increased its content in pbmc after the first acute bout in both groups . However, this upregulation was blunted by the training program after the second acute bout . Taken together, these findings support the role of the eccentric training as a valuable asset to decrease the inflammation associated with acute exercise . Thus, upon stimulation, tlr4 is internalized into endosomes where two transcriptional events occur: (1) delayed activation of nf-b and (2) initiation of the irf3 transcription program . In the latter case, trif activates two ikk related proteins through traf3, inducible ikk (ikki or ikk), and tank - binding kinase1 (tbk1). Ikk and tbk1 activate interferon regulatory factors, such as irf3, which dimerise, translocate to the nucleus, and bind interferon response elements (isre), resulting in the induction of type-1 interferons [30, 33]. In the present study trif protein levels increased after the acute eccentric exercise in parallel to an activation of ikk, and both effects were inhibited following 6 weeks of submaximal eccentric training . However, our data indicate a nonsignificant increase immediately after and at 2 h after the acute bouts in the irf3 protein . First, we cannot exclude the existence of a methodological limitation since data in the literature show that other authors used a phospho - specific antibody against ser386 to demonstrate that overexpressed irf3 is phosphorylated on ser386 and that phosphorylation on ser386 is observed in the dimeric form of irf3 [37, 38]. However, phosphorylation on ser396 (antibody used in this study) alleviates autoinhibition to allow interaction with cbp (creb - binding protein) and facilitates phosphorylation at ser386 . Second, tbk1 and ikki can also phosphorylate and activate irf7, which is the member of irf family most closely related to irf3 . Whereas irf3 is ubiquitously expressed and not inducible, irf7 is expressed at low levels in most types of cells but is strongly induced in response to various stimuli . It has been shown that the expression of some downstream effectors of the tlr signaling pathways, such as irf7, was reduced by addition of resveratrol to exercise training in rat heart . Furthermore, irf7, which is mainly activated by tlr7 and 9, seems to be the master regulator of type - i interferon - dependent immune responses and exerts transcriptional control over a large set of proinflammatory genes . The activity profile of the tlr4 pathway may be affected by exercise - induced changes in the pbmc subpopulations . In this study, there were no differences in the distribution of pbmc subpopulations between the first and the second acute eccentric bouts within a group or between groups . Therefore, differences in tlr4 signaling activity between tg and cg and between acute eccentric bouts can only be explained by the eccentric - based training intervention followed by tg . Additionally, it should be noted that none of the proinflammatory markers analyzed were downregulated in the cg . Thus, although the adaptations induced by a single bout of eccentric exercise may decrease the muscle damage from a subsequent bout (i.e., repeated bout effect), it appears that such adaptations do not affect the inflammatory response associated with eccentric exercise in young women . Indeed, the repeated bout effect failed to protect against the proinflammatory response in young men [6, 7]. In summary, this study provides evidences indicating that an acute eccentric exercise increases the tlr4-mediated proinflammatory response through both myd88- and trif - dependent pathways in young women, supporting previous reports in men . More importantly, we show that regular eccentric training may be an effective method to prevent the upregulation of some of the signaling pathways controlling the proinflammatory response in young women . This may be of great importance given the higher risks of women to suffer from autoimmune and inflammatory diseases . Further research assessing long - term effects of eccentric exercise on inflammatory profile in young women is therefore warranted . The present study also supports the notion that tlr4 signaling pathway is a key regulator of the exercise - induced inflammatory response, highlighting the role of tlr4 as a target for future exercise - based anti - inflammatory interventions . However, more research is needed to fully evaluate the potential associations between exercise, tlr4, and activation of the irf transcription factors . The current study provides novel understanding of the molecular mechanisms behind the inflammatory response controlled by the tlr4 signaling pathway after acute exercise in trained and untrained young women . It is suggested that different signaling pathways involving ikk and the transcription factors irf are modulated by tlr4 in response to exercise . Additionally, the eccentric training is purported to be a valuable tool to decrease exercise - induced inflammation through a downregulation of the tlr4 downstream signaling in young women . In contrast, the repeated bout effect does not seem to modify the eccentric exercise - induced inflammatory response in women . These data may have implications in the prevention and rehabilitation programs currently employed for autoimmune and inflammatory diseases in this population.
Systemic lupus erythematosus (sle) is a chronic systemic autoimmune disease with considerable heterogeneity in clinical manifestations and disease course . The etiology of sle remains unknown, but evidence suggests that sle results from the interaction of genetic and environmental factors that ultimately promote an abnormal immune response leading to the organ damage . Dysregulation of various components of the immune system can be seen at different stages of sle development, but hyperactivity of b cells with excessive production of multiple autoantibodies is perhaps the major immunological stigmata of sle [2, 3]. The pathogenic role of b cells in sle is not however strictly linked to the secretion of autoantibodies . B cells can have potent antigen presentation capacity, and they regulate and organize inflammatory responses . Indeed, studies in murine models of sle have shown that b cells are critical to the development of disease even when they are unable to secrete autoantibodies [5, 6]. Moreover, studies in human sle showed that the clinical response obtained by treatment of patients with rituximab, a b cell depleting anti - cd20 antibody, preceded autoantibody decline, thus supporting the autoantibody - independent role of b cells in sle process . A more detailed analysis of molecular mechanisms underlying the sle - associated tissue damaging - immune response revealed that interactions between t and b cells are essential in the development of this disease and that cytokines are key mediators of this interplay . However, because of the redundant functions of cytokines, a major challenge for scientists is to identify key molecule(s) which can profoundly alter the outcome of sle - associated inflammatory response . Here although il-21 was initially considered as a cytokine produced by activated peripheral blood t cells, it is now known that it can be synthesized by a range of differentiated cd4 + t helper (th) cells . Studies in mice have shown that il-21 is synthesized exclusively by polarized th17 cells, while in human, il-21 can also be made by th1 cells [1116]. More recently, it has been shown that il-21 is also produced by a subset of cd4 + t cells, which are involved in germinal center (gc) formation and function, and denoted t follicular helper cells (tfh cells) [18, 19]. Tfh cells express high levels of the chemokine receptor chemokine (c x c motif) receptor 5 (cxcr5), which allows them to home to and be retained by the lymphoid follicle, where contact with antigen - primed b cells promotes b cell proliferation, isotype switching, and somatic mutation of the ig repertoire [20, 21]. . However, these cells can synthesize il-21 if deleted of specific transcription factors, such as t - bet and eomesodermin, and infected with lymphocytic choriomeningitis virus . Il-21 activity is mediated by a heterodimeric receptor, formed by the -chain subunit (shared with il-2, il-4, il-7, il-9, il-13, and il-15 receptors) and its own unique receptor (designated il-21r) [23, 24]. Binding of il-21 to the receptor leads to the activation of the janus - kinase - family proteins (jak 1 and jak 3) and signal transducer and activator of transcription (stat) 1 and stat3 and, to a lesser degree, stat4 and stat5 . Il-21 also activates members of the mitogen - activated protein kinase (mapk) family such as extracellular signal - regulated protein kinases (erk) 1/2 in neoplastic cell, epithelial cells, and monocytes [25, 26]. Strictly linked to the activation of such intracellular pathways is the ability of il-21 to regulate the functional activity of various immune and non - immune cell types . Here we highlight some of the biological effects of il-21 on b and t cells, because of the pathogenic role of these cells in sle . For a detailed description of the regulatory functions of il-21 on both immune and non - immune cells, the reader is directed towards recent reviews [10, 23, 24, 27]. Il-21 increases b cell proliferation after activation via the b cell receptor plus t cell - derived costimulatory signals [28, 29]. In contrast, il-21 delivers proapoptotic signals to primary b cells and b cells that are activated with anti - igm or toll - like receptors ligands [30, 31]. In cultures of cd40-stimulated human b cells, the proliferative effect of il-21 is dependent on stat1 and stat3 activation . In vitro, stimulation of both murine and human b cells with il-21 induces the plasma - cell - associated transcription factor b - lymphocyte - induced maturation protein 1, now termed pr domain containing 1, and subsequent plasma cell differentiation as well as the production of isotype - switched immunoglobulins (i.e., igg, igm, and iga) [28, 29, 33]. Importantly, the il-21-driven generation of memory b cells and plasma cells relies on stat3, because il-21 fails to promote the differentiation of ig - secreting cells in cultures of nave b cells isolated from patients with inactivating mutations in stat3 . Using a well - established model of b cell differentiation, avery et al . Have also shown that the hen egg lysozyme - specific response of il-21r - null b cells is diminished compared with wild - type b cells, as indicated by the marked reduction in the number of antigen - specific plasma cells and production of igm and igg . Overall, these results suggest that il-21 and stat3 play a crucial role in initiating and/or maintaining serological immunity by generating antigen - specific effector b cells from nave precursors . By contrast, stat3 does not seem to be necessary for the il-21-driven isotype switching to igg . Il-21 enhances the proliferation of anti - cd3-stimulated t cells and acts in concert with other common -chain - dependent cytokines to enhance the growth of cd4 + t cells [10, 24]. However, il-21 regulates differently the differentiation and/or maintenance of polarized th cells depending on the species . Studies in mice revealed that il-21 can expand either th2 or th17 cell responses and inhibit the synthesis of interferon (ifn)- [11, 12, 3436]. By contrast, in populations of human peripheral blood or mucosal t cells that are preactivated with anti - cd3, il-21 enhances the expression / activation of transcription factors that drive th1 cell differentiation (i.e., t - bet, stat4) [14, 37]. Il-21 stimulates the proliferation of cd8 + t cells and synergizes with il-15 and il-7 in promoting cd8 + t cell expansion [3840]. Studies in il-21-deficient mice showed that cxcr5 surface expression on cd4 + t cells is greatly reduced after immunization with a t - cell - dependent antigen, and that il-21r expression is significantly higher on cxcr5+cd4 + than on cxcr5cd4 + t cells . Adoptive transfer of wild - type cd4 + t cells into il-21r - null recipients followed by immunization rescues gc formation and partially rescues ig production . The fact that il-21 controls the pool of memory b cells and promotes differentiation of b cells into plasma cells suggests that a deregulated il-21 activity may contribute to the development of autoimmune diseases . So, many researchers have evaluated the contribution of il-21 in the pathogenesis of murine models of sle these strains result from a cross between a c57bl/6 female and an sb / le male, and the male offspring of the cross had a 50% mortality rate at 6 months of age . The mice display many of the symptoms common to sle, including lymphadenopathy, splenomegaly, hypergammaglobulinemia, and severe immune complex mediated glomerulonephritis . Subsequent studies have verified that the disorder is not gonadal hormonally driven but is y - linked . Analysis of multiple genes in splenocytes taken from these mice revealed a marked age - dependent increase in the levels of il-21 mrna as compared to wild - type mice . Corresponding to the increase in il-21 mrna, serum levels of il-21, igg1, and igg3 were increased in bxsb.b6-yaa+/j mice . Importantly, il-21r - deficient bxsb - yaa+/j mice show none of the abnormalities characteristic of sle, thus supporting the key role of il-21 in the accumulation of plasma cells and production of autoantibodies . In this model, the excessive il-21 production did not derive from tfh cells, but rather from an extrafollicular population of icos+ cd4 + t cells . Further support to the pathogenic role of il-21 in this model of sle was provided by preclinical studies showing that administration of il-21r / fc, a fusion protein neutralizing il-21, to bxsb.b6-yaa+ mice results in a decreased production of il-21, diminished lymphocyte activation, and reduced circulating igg1 levels . Proteinuria is also reduced in treated mice, but the therapy does not prevent the symptoms of sle . Moreover, follow - up studies showed that the il-21 contribution to sle - like phenotype in bxsb - yaa mice is variable within the time course of disease progression, because blockade of il-21 activity in the early phase is deleterious, whereas later in the time course it is advantageous . The reason why the blocking il-21r / fc regulates differently the pathogenic inflammatory response in bxsb - yaa mice remains unknown . In this context, it is noteworthy that il-21 can exert both inflammatory and anti - inflammatory effects, the latter linked to the induction of il-10, a counter - regulatory cytokine expressed at high levels both in bxsb - yaa mice and in human sle patients [33, 4547]. Therefore, blockade of il-21 with il-21r / fc might inhibit il-10 expression, thus exacerbating the severity of sle symptoms in the early phase of the disease . Studies in mrl - fas mouse, another model of sle, showed that blockade of il-21 with il-21r / fc significantly reduced proteinuria, lymphadenopathy, skin lesions, circulating autoantibodies, and igg1 and igg2a . In addition, mrl - fas mice treated with anti il-21r / fc showed reduced levels of glomerular igg deposits in the kidney and no thickening in glomerular basement membranes by histological evaluation . Il-21r / fc treatment also reduced the number of splenic t lymphocytes and b cells antibodies production . In the mrl - fas mouse, il-21 is primarily made by an extrafollicular population of icos - expressing cd4 + t cells that exhibits reduced expression of p - selectin glycoprotein ligand 1 but is able to produce il-4 and ifn- . Evidence for the pathogenic role of il-21 in sle also comes from studies in the sanroque mouse strain, in which a mutation in the ring - type ubiquitin ligase protein family member, roquin, results in the accumulation of excessive numbers of both gc and tfh cells with high levels of icos, excessive il-21 production, and severe sle - like autoimmune phenotype . Lupus - like symptoms are dependent on enhanced gc formation as they could be reduced by deletion of even one allele of the bcl6 gene . However, tfh formation in this model seems to be dependent on icos rather than il-21 . A subpopulation of b-1 cells expressing the programmed death ligand 2 (termed l2pb-1 cells) has been shown to be enriched for autoreactive immunoglobulin, to be potent in antigen presentation, and to be fully able to facilitate th1 and th17 cell differentiation . L2pb1 cells are found in increased numbers in the periphery of mice with sle and such an increase correlates with anti - dna antibody levels . This well fits with the demonstration that l2pb-1 cells secrete high levels of autoantibodies, such as dsdna - reactive antibodies, and can undergo isotype switch and generate igg1 and igg2b in the presence of il-21 . Plasma levels of il-21 are significantly higher in sle patients than in healthy controls, but il-21 levels do not seem to correlate with sle activity . We have recently shown that il-21 mrna expression is enhanced in skin biopsies taken from individuals with sle in comparison to normal controls, even though this increase is less pronounced than that seen in lesional skin of patients with psoriasis . In patients with sle, il-21r is under - repressed on total, nave, and memory b cells, and on plasmablasts, compared to controls . Reduced levels of il-21r are associated with nephritis and a high titer of anti - double - stranded dna antibodies in the serum . By contrast, blood t cells of sle patients exhibit normal levels of il-21r . Recent genome - wide association (gwa) studies have shown that single nucleotide polymorphisms (snps) in the chromosome 4q27 region containing il-2 and il-21 are associated with chronic inflammatory disorders, including inflammatory bowel disease (ibd), celiac disease, psoriasis, diabetes, rheumatoid arthritis, asthma, and sle, suggesting a common genetic background for these diseases [5662]. With regard to sle, a first study genotyped three snps in the il-21 gene in more than 1300 patients with sle and controls . Two out of the three snps (rs907715 and rs2221903) were significantly associated with sle . Stratification by race revealed that this genetic association was found more in european - american than in african - american sle patients . The fact that both associated snps are located within intronal regions of the il-21 gene suggests however the possibility that they can influence il-21 production . Another gwa study analyzed 17 snps in the il-21r gene in 2 independent, ethnically divergent populations (european and hispanic) of sle patients and controls . A genetic association between rs3093301 (a / g) and sle was found in both the european - derived and hispanic cohorts . Moreover, the lupus - risk allele was significantly associated with the presence of malar rash but not other symptoms or signs of sle . The involvement of il-21 in the pathogenic mechanisms causing human sle remains to be demonstrated . However, both the ability of this cytokine to regulate in vitro and in vivo immunological pathways that are relevant in sle and the evidence obtained in murine models of sle suggest that il-21 could play an important role in the production of pathogenic autoantibodies and end - organ damage in this disease . Therefore, it is tempting to speculate that inhibitors of il-21 could be useful to attenuate lupus - related clinical manifestations . However, in designing such clinical interventions for blocking il-21 one should take into consideration not only the advantageous effects but also the risk of potential and deleterious consequences for the host . In fact, it is well - known that, at least under specific circumstances, il-21 can also exert anti - inflammatory actions due to its ability to inhibit dendritic cell maturation and stimulate il-10 production [45, 64]. In this context, it is also noteworthy that il-21 may stimulate immune responses against tumor cells and promote cd8 + t cell responses against viruses [6568]. Therefore, if so, blockade of il-21 function could attenuate antineoplastic immunity and wake worse the course of viral infections.
Peripartum depression is the most frequent form of maternal morbidity in pregnancy and affects 15%20% of mothers in the first year following delivery; 10% of fathers are also affected . Approximately 18% of women exhibit depressive symptoms antenatally . In 2013, the american psychiatric association amended the nomenclature postpartum depression to peripartum depression and stipulated that the onset of depression can occur in pregnancy or after childbirth . Peripartum depression affects the individual s quality of life; its impact extends to the partner and family and influences mother multiple sclerosis (ms) is a chronic condition of the central nervous system, with clinical symptoms characteristically manifesting in early adulthood aged between 20 and 40 years, when individuals are of reproductive age . Depression and anxiety affect individuals with ms at twice the frequency observed among people without ms . Although many individuals with ms will experience depression, there is a dearth of information on the rate of peripartum depression among parents with ms . Maternal postnatal psychological distress is a significant predictor of childhood anxiety even after controlling for distress in the prenatal and early childhood periods . Previous work has demonstrated that parents with ms have a substantially higher rate of mental health disorders as compared with parents not affected by ms . Also, children of a parent who has both ms and a coexisting mental health condition are at an increased risk of developmental problems . We, therefore, carried out a study to examine the timing of parental depression, specifically peripartum depression, and its potential association with psychiatric disorders among children exposed to parental ms since birth . This study was part of a broader programme of research examining the association between parental ms and routinely collected childhood developmental outcomes . The publicly funded provincial health care programme in british columbia covers all residents; a lifelong unique personal health care number is assigned to each resident and available in all health databases . The anonymized linked health data files used in this study included the discharge abstract database (with hospital admission dates and diagnosis codes), the medical services plan billing database (providing information on physician visits and diagnosis codes), the pharmanet database (with information on dispensed prescriptions) and vital statistics birth files (with records of all births in the province). Registration files provided dates of entry and exit from the provincial health care plan (which confirmed residency in british columbia), and socioeconomic status (ses) was based on average neighbourhood income (obtained through postal codes and national census data), expressed as quintiles . Diagnoses in these databases were coded using the international classification of diseases codes (icd-9 or icd-10-ca), and prescription medications were coded using drug identification numbers and the anatomical therapeutic chemical (atc) classification system . Ms in parents was identified using a validated algorithm, as those with 3 records related to ms in hospital admission or physician visit claims between 1 april 1985 and 31 december 2011 or in prescription claims after 1 april 1996 (supplementary appendix table 1). Parents with ms were linked to their offspring using the birth registry and the registration file databases . All persons with ms who had a child born in british columbia between 1 january 1994 and 31 december 2006 were included in the study cohort . The ms cohort was restricted to individuals whose ms onset occurred before their child s birth, based on the first date for ms or a demyelinating condition identified in any of the hospital, physician or prescription claims (supplementary appendix table 1). A matched reference cohort of up to four children with parents who were not known to have ms or a demyelinating condition was selected from the population of british columbia . Children in the matched reference cohort were matched on the year of birth and school district . The parent with ms was also matched by sex to a parent in the reference cohort . In instances where exact matching for parental sex could not be carried out (i.e. In 4% of cases), the available parent in the database was selected . All children in the study were followed up for a minimum of 4 years and up to 18 years between 1994 and 2011 . Peripartum depression was defined to include mood or anxiety disorders since these conditions have a shared psychopathology, are highly comorbid and are often difficult to distinguish in primary care . Parents in the study were classified as having peripartum depression if they had one or more records related to mood disorders or anxiety in hospital, physician or prescription drug claims in the last 4 weeks before delivery and up to 12 months after the child s birth (supplementary appendix table 1). Subsequent parental depression or anxiety (in the period following the peripartum period) was identified based on one or more records related to mood disorders or anxiety in hospital, physician or prescription drug claims after the peripartum period . History of depression was defined as the presence of depression or anxiety in the 2 years prior to the peripartum period . Psychiatric disorders in children were defined based on one or more records for internalizing (mood or anxiety) psychiatric disorders or externalizing (attention - deficit hyperactivity disorder (adhd) or conduct) psychiatric disorders in physician or hospital claims (supplementary appendix table 1). The date associated with the first record of such a psychiatric disorder was considered the date of onset for the condition . Diagnosis of a psychiatric disorder in children was restricted to the period after the child s fourth birthday since the accurate diagnosis of depressive and anxiety disorders in very young children is often difficult due to issues related to cognitive and emotional immaturity, and the asymptomatic expression of depression in children . Although recent studies have used a similar approach to identifying mental health disorders, we also identified mental health disorders in children and parents using a previously validated algorithm . The latter diagnoses were used in a sensitivity analysis to assess whether results comparing children of parents with and without ms were affected by potential changes in diagnostic accuracy . The date of cohort entry (index date) for all children was defined as the month of the child s fourth birthday . Children were followed up from the index date until the first diagnosis of a psychiatric disorder, emigration from british columbia or the study end date, which was 31 december 2011 . The child s characteristics examined included sex, the child s first language at home (english vs other), presence of an older sibling (yes vs no) and ses at index date (expressed as quintiles). The parental characteristics studied included parental sex, parental age (continuous) and marital status at the time of the child s birth . Conditional logistic regression was used to compare the characteristics of the parent child dyads in the ms and matched reference cohorts . The frequency of peripartum depression among parents with and without ms was estimated using cumulative rates and 95% confidence intervals (cis). Multivariable log - linear regression models with robust variance estimators were used to determine the association between parental ms and peripartum depression, after adjusting for potential confounders including parental marital status, parental subsequent depression or anxiety, age at the time of the child s birth, parental sex and ses . The incidence (density) of psychiatric disorders in children among parents with and without ms was quantified by dividing the number of children with psychiatric disorders by the total follow - up time in child - years in each category . Incidence patterns of psychiatric disorders in children were examined using kaplan meier curves and cox proportional hazard regression accounting for matched factors . In the cox models, standard errors were adjusted for within - family clustering to account for the sequential births to the same parent in the cohort . Confounders were included in the final model based on the literature or statistical significance (p value <0.1), and the proportionality assumption in the cox model was checked for each variable in the model . The full cox model included the child s sex (female vs male), ses (expressed as quintiles), peripartum depression (present vs absent), parental subsequent depression or anxiety (present vs absent) and parental marital status (not married vs married). Modification of the effect of parental ms by other factors was examined using interaction terms, and stratified analyses were presented to illustrate potential effect modification . Results were expressed as odds ratios (ors) and hazard ratios (hrs) with 95% cis . Regression model fit was assessed using the likelihood ratio test, and a two - sided p value <0.05 was used to determine statistical significance . Analyses were performed using sas version 9.2 (sas institute inc ., cary, nc). The study cohort included 360 children with an ms parent and 1207 parent child dyads in the matched reference cohort (figure 1). The characteristics of children with a parent with ms and those with unaffected parents were similar in terms of sex, birth order and ses at cohort entry (table 1). Parents affected by ms were on average older, more likely to be english speakers, have a higher frequency of subsequent depression and to be married at the time of the child s birth . The median age at onset of ms was 28 years and the median disease duration at the index date was 8.5 years . Approximately 32% of the parents affected by ms had received disease - modifying medication by the index date (table 2). Characteristics of parents with and without multiple sclerosis (ms) and their children, british columbia, canada . From conditional logistic regression that accounted for matching at the design stage . Additional characteristics of the cohort of parents with multiple sclerosis (ms), british columbia, canada . N=30 (26%) were exposed to glatiramer acetate and n= 85 (74%) to a beta - interferon . Parents affected by ms had higher rates of peripartum depression compared with parents in the reference cohort (25.8% vs 18.5%, p value 0.004; table 1). Similarly, fathers who had ms had considerably higher rates of peripartum depression compared with unaffected fathers (25.7% vs 10.2%; figure 2). Among individuals with peripartum depression, 52% of parents with ms and 48% of unaffected parents had a prior history of depression (supplementary appendix table 2). Adjusted analyses showed that parental sex modified the effect of parental ms on peripartum depression (p value for interaction term 0.008; table 3). Adjusted analyses stratified by parental sex showed that the rate of peripartum depression was 2.68 times higher (95% ci: 1.684.29) among fathers with ms as compared with fathers not affected by ms, while the rate of peripartum depression was 28% higher (95% ci: 0.991.65) among mothers with ms as compared with mothers without ms . Peripartum depression (%) by parental multiple sclerosis (ms) status and parental sex, british columbia, canada . Unadjusted and adjusted odds ratios and 95% confidence intervals from log - linear regression showing the association between parental multiple sclerosis (ms) and peripartum depression, british columbia, canada . Unadjusted model with matching factors (child s year of birth, school district and sex of the parent). Adjusted model with matching factors (child s year of birth, school district and sex of the parent) plus variables listed in the table and an interaction term (parental ms * parental sex: unexponentiated beta 0.65; 95% ci: 0.131.18). During the follow - up period, 226 children (14%) were diagnosed with a psychiatric disorder (table 1). Among children with psychiatric disorders, mood or anxiety disorders were more common than adhd and conduct disorders . In the kaplan meier analysis, 1309 (83.5%) children were followed up until the end of the study period, 226 (14.4%) until the diagnosis of a psychiatric disorder and 32 (2.1%) until emigration from the province . The incidence of psychiatric disorders in children aged 417 years (per 100 child - years) was 3.3 among children of parents with ms and 2.7 among those with parents not affected by ms (crude hr: 1.19; 95% ci: 0.931.54; table 4). The incidence rate of psychiatric disorders in children was higher among boys, children whose parents had peripartum and subsequent depression and children whose parents were not married (table 4). Cox regression that accounted for matching at the design stage) the adjusted cox model showed that children with parental ms had a 34% higher rate of psychiatric disorders compared with children without parental ms (adjusted hr: 1.34; 95% ci 1.031.74; table 5, model 2). The rate of psychiatric disorders was also higher among children whose parents had peripartum depression compared with children whose parents did not have peripartum depression (adjusted hr: 1.87; 95% ci 1.32.55). Model 2 further showed that the relationship between parental ms and peripartum depression and child psychiatric disorders remained statistically significant even after controlling for subsequent parental depression or anxiety . Peripartum depression did not modify the effect of parental ms on psychiatric disorders in children (p value for interaction term 0.25). Sensitivity analyses based on mental health disorders identified using a previously validated algorithm yielded similar results . Adjusted hazard ratios and 95% confidence intervals from cox regression showing the association between parental multiple sclerosis (ms) and psychiatric disorders in children, british columbia, canada . Model 1: adjusted model with matching factors (child s year of birth, school district and sex of the parent) plus remaining variables listed in the table . Model 2: model 1 plus subsequent parental depression or anxiety after the peripartum period . Sensitivity analyses carried out to ascertain whether the higher rate of peripartum depression among fathers with ms was secondary to their increased contact with the health care system showed that fathers with ms had an average of 11 physician visits during the peripartum period, while fathers without ms had 7 visits . The association between ms and peripartum depression among fathers was attenuated but remained significant even after adjusting for physician visits (rate ratio: 1.96; 95% ci 1.143.40). Our study provides evidence that parents with ms had an increased risk of peripartum depression as compared with parents unaffected by ms . Furthermore, children of parents with ms had a higher risk of developing psychiatric disorders compared with the children of parents without ms . Similarly, children whose parents had peripartum depression had a higher risk of developing psychiatric disorders compared with children of parents without peripartum depression . Higher rates of psychiatric disorders in children were also observed among boys, children whose parents had subsequent depression or anxiety and those who were not married . To the best of our knowledge, there have been no previous reports that have quantified the frequency of depression or anxiety in the peripartum period among parents with ms . Studies in women with epilepsy show that approximately 25%29% of such women screen positive for depression . In our study, approximately 26% of mothers with ms were identified as having peripartum depression or anxiety, compared with a lower 21% rate in mothers without ms . Depression is frequently missed among patients with ms and even when detected is often inadequately managed . Maternal depression is particularly concerning because it is often a risk factor for a poor quality of life, paternal depression and adverse emotional, intellectual and cognitive development in children . Peripartum depression in people with ms could also adversely impact an individual s adherence to treatment, and this can have important consequences on their disease course . In our study, fathers with ms had substantially higher rates of peripartum depression or anxiety (26%) compared with fathers without ms (10%). The latter rate is consistent with studies which show that peripartum depression is not uncommon among fathers and has a 12-month postpartum prevalence of 4%10% . An elevated risk of depression in those with ms compared with those without ms has also been observed in other studies; rates of depression among women with ms are 59% higher than among women without ms, while among men, rates of depression are 93% higher in those with ms compared with those without ms . The higher risk of peripartum depression in men with ms could be triggered by their new role as fathers, which may require them to confront their physical or cognitive impairments and ability to address their family s emotional and financial needs . Furthermore, ms - related fatigue and physical disability and the unpredictable nature of ms symptoms can affect their ability to play an active role in caring for their young children and threaten their self - expectations of fatherhood . The association between parental ms and psychiatric disorders in children is consistent with previous work demonstrating that maternal ms is associated with a higher rate of mood or anxiety disorders in children; this association appears to be mediated through maternal mental health morbidity . The limited research on this issue supports the notion that children of parents with ms are at greater risk of psychiatric outcomes including higher levels of depression and anxiety as compared with the children of healthy parents . Our study shows that both peripartum depression and subsequent parental depression independently increase the risk of psychiatric disorders in children . Other studies have also demonstrated that maternal postnatal psychological distress is a significant predictor of childhood anxiety even after controlling for distress in the prenatal and early childhood periods . Studies examining the role of parental depression on child mental health suggest that the rates of psychiatric disorders among children of depressed parents are two to five times above normal and that the risk associated with maternal depressive symptoms may be comparable with that of paternal depressive symptoms . Timely and appropriate interventions are key for such families; intervention studies targeted towards treatment for parental depression have shown significant improvement in children s functioning and psychiatric symptoms within 1 year after initiation of treatment . The strengths of our study include use of a comprehensive population - based data source and the use of previously validated case definitions for ms . The robustness of our analyses was also confirmed by the supplementary analyses in which psychiatric disorders were identified using a previously validated algorithm . We also accounted for the clustered nature of the data arising from sequential births to the same parent during the study period . One limitation of our study was our inability to assess severity of peripartum depression which could have differed between mothers and fathers . Furthermore, the time of onset of peripartum depression was defined as the date of the first record for a mood or anxiety disorder during the peripartum window, which may not necessarily represent the date of onset of symptoms . It is possible that more frequent contact with medical services in our ms cohort increased the opportunity to get diagnosed with a mental health disorder . However, adjustment for health services use did not abolish the association between paternal ms and peripartum depression . Another limitation of our study arose because the study sample was restricted to children born between 1994 and 2006, who had childhood developmental data . However, given that the assessment of childhood developmental outcomes has been routinely administered province - wide, the generalizability of our findings to the wider population of parents with ms was likely unaffected . Finally, we did not have a family - specific variable to assess ses, but used neighbourhood - level income as a proxy for ses . Our study demonstrated that parental ms, specifically paternal ms, is associated with a higher risk of peripartum depression . Furthermore, parental ms and parental peripartum depression independently increase the risk of psychiatric disorders in children . Given that depression and other psychiatric disorders are believed to be under - diagnosed and under - treated in persons with ms, parents with ms require special attention from health care professionals to ensure that their mental health and their children s mental health are optimized.
The first report of vaginal evisceration was described by hyernaux in 1864, as disruption of the anterior wall of the proximal vagina, resulting in prolapse of the abdominal contents (1). Since then, there have been just over 120 reports in the literature, although in reality some cases may go unreported (1). Vaginal evisceration is a rare postoperative complication of hysterectomy (regardless of the surgical approach) in women of this age group that carries a high risk of morbidity and mortality and requires rapid surgical intervention . There are different aetiologies depending on the age group pre - menopausal vs postmenopausal (2). In post - menopausal women interestingly, in pre - menopausal women there have been reports of vaginal evisceration following transvaginal ultrasonography (3). The use of each approach depends on the viability of the bowel, whether the defect in the vaginal wall is sufficiently large to replace the bowel back into the peritoneal cavity and on any evidence of foreign bodies in the abdominal cavity . A 66 year old woman presented to a&e with per vagina bleeding, abdominal pain and a mass protruding from the vagina . Prior to this presentation, the patient had suffered for the past decade with cystocoele and retrocoeles . Three decades ago the patient had received an abdominal hysterectomy in order to relieve her symptoms of menorrhagia . Other than arthritis, the patient does not suffer from any other medical condition and was in good health . On examination the patient seemed well, but her abdomen was distended and tender in the suprapubic, left and right iliac fossa regions . There was evidence of vaginal prolapse with a 20 - 30 cm evisceration of small bowel via a vaginal vault defect . Prior to the emergency operation which aimed to salvage the small bowel, the patient was given antibiotics . The aim of the surgery was to reduce the prolapsed small bowel and re - suture the vaginal wall defect . On inspection by the consultant gynaecologist, the small bowel appeared slightly dusky and an opinion was sought from a consultant general surgeon (who deemed the bowel healthy). The small bowel was replaced through the vaginal wall defect and vaginal wall biopsies taken . The patient recovered well in the first few days after the operation, though she did have some trouble maintaining the catheter in situ, due to the still uncorrected vaginal prolapse . The catheter was removed on day 5 post - operatively, but the patient had developed a urinary tract infection while catheterised and had to be on i.v . The patient was discharged on day 8, but was to come back 6 - 8 weeks later for an elective sacrocolpopexy to correct the vaginal wall prolapse . Initially exploratory laparoscopy was performed, the pelvis appeared normal and the consultant decided to perform an abdominal sacrocolpopexy . A pfannenstial incision was made, small bowel adhesions to right pelvic side wall were noted and divided . The peritoneum between sacrum and posterior vaginal wall was exposed, allowing prolene mesh to be attached to the vaginal wall with prolene and vinyl sutures . The rectus sheath was sutured using vinyl and the skin closed with staples (with a drain in situ). Cystoscopy was used to verify the function of the ureters, which revealed both were working well . The patient recovered quickly, there were no post - operative complications and was discharged on day 2 post - operatively . This patient had had a hysterctomy many years ago and it is unlikely her vaginal evisceration was a consequence of this surgery directly, but hysterectomy can lead, for example to changes in the vaginal axis . The vagina may come to lie in a more descended position, as it has lost part of its more proximal attachments . This new position combined with factors such as cystocoeles and retrocoeles and their management (for example the insertion of pessaries) may predispose the vagina walls to more wear and tear damage . These are risk factors that are likely to have contributed to this lady s transvaginal evisceration, together with her increasing age susceptibility . Vaginal evisceration if not recognised quickly can lead to peritonitis and bowel ischaemia . In this lady however, it is important to make emergency doctors aware of these complications and encourage their proper management . When the patient is received by an emergency department and the problem is determined, the emergency doctors must not delay the administration of broad spectrum antibiotics in preparation for taking the patient to theatre and ensure the prolapsed bowel is covered in saline - soaked gauze, as this improves the bowel chances of viability (4). This case illustrates the need for rapid recognition of vaginal evisceration and its appropriate management in order to avoid morbidity and mortality to the patient.
The new proposal for classification of focal cortical dysplasia (fcd) recognizes a particular type, iiid fcd, associated with an epileptogenic lesion acquired in early life (i.e., traumatic injury, ischemic injury or encephalitis). Fcd group associated with certain other epileptogenic lesions (in particular, hippocampal sclerosis, tumors and vascular malformations). Histopathologically, type iiid fcd consists of altered cortical architecture and cytoarchitectural composition, occurring adjacent to the principal early - life lesion . There is not yet much information regarding the prevalence of these cortical dysplastic changes among patients with these kinds of early - life lesions, and imaging pathologic correlations are only at the beginning; nevertheless, it could be inferred that such an association is probably high . Epileptic seizures secondary to early - life brain injuries often follow a protracted and drug - resistant course . Given the destructive nature and involvement of multiple brain regions by initial injury, many of these patients are not considered suitable for epilepsy surgery . Others with well - lateralized hemispheric lesions undergo extensive procedures (hemispherectomy / hemispherotomy) which, although associated with successful postsurgical seizure control, may induce new or aggravate previously existing neurological deficits . This work aims to denote that a) selected cases with converging semiological, electrophysiological and structural / functional imaging evidence of localized ictal onsets may show favorable postsurgical results to focal - tailored resections and b) accompanying mesio - temporal pathology ipsilateral to the gross lesion may not contribute to seizure generation . Patient 1 was a 10-yr - old boy originally diagnosed with left hemispheric atrophy and an ipsilateral parietotemporooccipital porencephalic cyst . He developed right - sided upper extremity hemiparesis, mild left superior quadrantanopia, and drug - resistant seizures (for history, presurgical evaluations and treatment details, see the supplementary data). Recent mri verified the presence of left hemispheric atrophy and large left - sided parietotemporooccipital porencephalic cyst and revealed an ipsilaterally malformed hippocampusmedial temporal region with increased flair signal (fig . An ipsilateral multilobar, parieto - occipital resection, sparing the mesial temporal structures, rendered the patient seizure free (fig . Patient 2 was a 25-yr - old man with lifelong history of intractable seizures secondary to serious posterior left head trauma sustained at the age of 4 months . Magnetic resonance imaging (mri) presented posterior left hemispheric atrophy, a prominent area of tissue loss and gliotic lesion over the lateral temporoparietal region and ipsilateral hippocampal atrophy plus temporal horn enlargement (fig . 2a). Surgical treatment involved extensive resection of the lateral temporo - parieto - occipital area, including the principal gliotic region and the lateral - inferior occipital region, sparing the medial temporal area (fig . Patient 1 was a 10-yr - old boy originally diagnosed with left hemispheric atrophy and an ipsilateral parietotemporooccipital porencephalic cyst . He developed right - sided upper extremity hemiparesis, mild left superior quadrantanopia, and drug - resistant seizures (for history, presurgical evaluations and treatment details, see the supplementary data). Recent mri verified the presence of left hemispheric atrophy and large left - sided parietotemporooccipital porencephalic cyst and revealed an ipsilaterally malformed hippocampusmedial temporal region with increased flair signal (fig . An ipsilateral multilobar, parieto - occipital resection, sparing the mesial temporal structures, rendered the patient seizure free (fig . Patient 2 was a 25-yr - old man with lifelong history of intractable seizures secondary to serious posterior left head trauma sustained at the age of 4 months . Magnetic resonance imaging (mri) presented posterior left hemispheric atrophy, a prominent area of tissue loss and gliotic lesion over the lateral temporoparietal region and ipsilateral hippocampal atrophy plus temporal horn enlargement (fig . 2a). Surgical treatment involved extensive resection of the lateral temporo - parieto - occipital area, including the principal gliotic region and the lateral - inferior occipital region, sparing the medial temporal area (fig . Given the recent introduction of the new classification scheme for fcd, it is not surprising that there is not yet much information regarding the incidence of iiid fcd among patients with epilepsy with early - life - acquired lesions and corresponding surgical outcomes . It is very likely that this type of pathology eluded detection in previous years, the neuropathologic focus being the principal lesion . . Reported on a series of 25 children having suffered pre- or peri - natal (mostly ischemic) injury, all with a broad spectrum of accompanying cortical dysplastic changes, mostly type i fcd (which apparently conforms to type iiid current classification). Slightly more than half of these patients were offered lobar and multilobar resections with class i engel outcomes achieved in 50% of them . There was a high incidence of extensive and not well - localized interictal and ictal eeg abnormalities in this series, suggesting the presence of large and diffuse epileptogenic zones . A subgroup of eight cases had had an early - life - acquired anoxic ischemic or inflammatory lesion . All eight patients underwent multilobar resections with class i engel outcomes achieved in only 25% of them . In contrast to the abovementioned studies, iida et al . Reported engel class i outcomes in 6/8 patients with congenital porencephalic cysts undergoing focal cortical resections guided by acute intraoperative ecog . Most of these patients had semiological evidence plus well - localized and lesion - concordant surface eeg findings in the presurgical work - up, implicating discrete cortical areas for ictal genesis . An interesting feature in both our cases was the mri presence of additional medial temporal pathology on the side of operation . This could be compatible with medial temporal sclerosis in patient 2, while patient 1 presented more complex features of hippocampal parahippocampal region white gray matter blurring, increased t2/flair signal and malformed / abnormally rotated hippocampus . These findings should raise the possibility of medial temporal ictal onsets, given literature data suggesting such a relationship in patients with congenital extratemporal porencephalic cysts and medial temporal sclerosis [79]. However, neither semiology nor surface eeg localization supported such a hypothesis . A subtemporal strip placed in patient 2 demonstrated only late involvement of the mediobasal temporal region during seizures . In addition, both patients enjoy excellent seizure control, 30 months and 18 months postoperatively, without having resected medial temporal structures . Among 29 patients with pre- or peri - natal hypoxia and low - grade fcd, all of these patients underwent extensive resections (mostly hemispherectomies) including medial temporal structures, so it is difficult to deduce the epileptogenic potential the lesioned medial temporal region might possess . Among 24 patients with congenital extratemporal porencephaly, burneo et al . Reported 9 with additional medial temporal sclerosis; an incidence very close to that of krsek's study . Five of these patients had ipsilateral temporal lobe ictal onsets and achieved class i engel outcomes following temporal lobectomy . However, 3 others had extratemporal ictal onsets, based on video - eeg findings . Ho et al . Reported evidence of medial temporal atrophy / sclerosis, based on mri hippocampal and amygdalar volumetry in 21 out of 22 cases with congenital porencephaly . In contrast, reported ictal onsets localized in the vicinity of congenital extratemporal porencephalic cysts with excellent results (engel class i) following tailored resections . . Concerning neuropathologic classification, the recent proposal does not offer a specific subcategory among type iii fcd for cases with the constellation of fcd, early - life injury and medial temporal sclerosis, or any other medial temporal malformative lesion, if present distant to the principal early - life lesion and neighboring dysplasia . Our findings, along with literature data, suggest that the organization of the epileptogenic substrate in type iiid fcd probably varies along a spectrum including on the one side cases with extensive ictal onset zones and non - satisfactory outcomes following focal resections and on the other side cases with well - localized ictal onsets and engel class i outcomes following tailored resections, even though mri may suggest more extensive and complex pathology . Within such complex imaging lesions, the epileptogenic potential is not necessarily uniformly distributed, and abnormal areas (such as the hippocampal - medial temporal region) may actually not contribute to ictogenesis, while in other cases be an essential component of it . The real challenge in presurgical evaluation is, therefore, to carefully determine which part, if any, of the whole abnormality is critical for seizure onsets . Our experience suggests that stereotyped focal - onset seizure semiology along with concordant localized interictal / ictal eeg and functional data is essential for identifying such candidates.
Surgery of the chin has been used for decades to achieve a balance of the lower third of the face . Horizontal osteotomy for chin augmentation is an old and established procedure while medpore has become popular in the recent years for facial skeletal augmentation . The present study has been undertaken to compare the advantages and disadvantages of medpore and osseous augmentation in genioplasty procedure . The study comprised 16 patients with retruded chin, attending the outpatient department (opd) of oral and maxillofacial surgery, faculty of dental sciences, c.s.m . Medical university, lucknow, india . Out of the 16 patients, 8 underwent medpore augmentation (group b) while the other 8 underwent osseous augmentation (group a). It contains those relations that are suitable for cases requiring advancement genioplasty and have been divided into soft tissue and skeletal relations [gonzales ullao, rickettes, steiner epker and fish, dipaolo, quadrilateral analysis and burstone, (cogs analysis)][figure 1]. Cephalometric hard and soft tissue points and associated reference planes lateral cephalometric radiographs were taken preoperatively and 1 week, 6 weeks and 3 months postoperatively in a standardized fashion with teeth in centric relation and the lips in repose (i.e. No obvious strain). Each cephalogram was traced on acetate paper and the following landmarks were located on each tracing . The horizontal reference axis was defined by registering on sella (s) orienting 7 inferior to s - nasion (s - n) plane . Pogonion line was taken to evaluate the deficiency of chin coupled with e - line of ricketts, 0-meridian of ulloa and facial contour angle was considered to obtain the millimeter deficiency present in chin preoperatively and changes postoperatively . Normally, the anterior projection of the mentum approaches approximately the vertical line from the subnasale in the male and slightly anterior to the vertical line from the nasion in the female . The soft tissue relationships of the chin to the lip and nose are very useful for evaluation of chin projection . When the esthetic plane is formed from the nose tip to the pogonion, the upper and lower lips both lie 12 mm posterior to the esthetic plane . The ideal relationship between the chin and the lips are the same as those for caucasians . The chin should rest slightly posterior to the lower lip, and the lower lip should rest posterior to the upper lip. [16] on frontal examination, the lower face is often subdivided into an upper one - third [extending between the subnasale and lip contact (stomion)], and a lower two third (measured between the stomion and mentum). In the indians, the upper third is slightly shorter and the lower two - third is slightly longer . Other things that need to be considered include the depth of the labiomental fold, the thickness of the chin pad, and the labiomental angle . Dissection was performed through a standard intraoral vestibular approach, where the paravestibular incision extending from first premolar on one side to the first premolar on the other, was given . The mental nerves were exposed bilaterally by dissecting posteriorly along the inferior border of mandible in the bicuspid region and mucoperiosteum was elevated . Three vertical marks were inscribed to avoid transverse deviation and rotation of the inferior segment . Osteotomy lines were present 45 mm below the apices of the cuspids and 34 mm below the level of the mental foramina, so as not to jeopardize these structures. [710] care was taken to achieve sharp and straight cuts without creating curves . After mobilization of the lower segment, the chin was moved to the predicted position . The overlying soft tissue envelop was also inspected from the outside every now and then such that there is free mobility of the soft tissues surrounding the implant . A chin dressing with mild compression was placed after the operation to reduce swelling, to assist in mentalis closure, and to prevent hematoma or seroma formation . The patients were kept on a complete liquid diet for a week and progressed to a soft and then regular diet afterward . It contains those relations that are suitable for cases requiring advancement genioplasty and have been divided into soft tissue and skeletal relations [gonzales ullao, rickettes, steiner epker and fish, dipaolo, quadrilateral analysis and burstone, (cogs analysis)][figure 1]. Cephalometric hard and soft tissue points and associated reference planes lateral cephalometric radiographs were taken preoperatively and 1 week, 6 weeks and 3 months postoperatively in a standardized fashion with teeth in centric relation and the lips in repose (i.e. No obvious strain). Each cephalogram was traced on acetate paper and the following landmarks were located on each tracing . The horizontal reference axis was defined by registering on sella (s) orienting 7 inferior to s - nasion (s - n) plane . Pogonion line was taken to evaluate the deficiency of chin coupled with e - line of ricketts, 0-meridian of ulloa and facial contour angle was considered to obtain the millimeter deficiency present in chin preoperatively and changes postoperatively . Normally, the anterior projection of the mentum approaches approximately the vertical line from the subnasale in the male and slightly anterior to the vertical line from the nasion in the female . The soft tissue relationships of the chin to the lip and nose are very useful for evaluation of chin projection . When the esthetic plane is formed from the nose tip to the pogonion, the upper and lower lips both lie 12 mm posterior to the esthetic plane . The ideal relationship between the chin and the lips are the same as those for caucasians . The chin should rest slightly posterior to the lower lip, and the lower lip should rest posterior to the upper lip. [16] on frontal examination, the lower face is often subdivided into an upper one - third [extending between the subnasale and lip contact (stomion)], and a lower two third (measured between the stomion and mentum). In the indians, the upper third is slightly shorter and the lower two - third is slightly longer . Other things that need to be considered include the depth of the labiomental fold, the thickness of the chin pad, and the labiomental angle . Dissection was performed through a standard intraoral vestibular approach, where the paravestibular incision extending from first premolar on one side to the first premolar on the other, was given . The mental nerves were exposed bilaterally by dissecting posteriorly along the inferior border of mandible in the bicuspid region and mucoperiosteum was elevated . Three vertical marks were inscribed to avoid transverse deviation and rotation of the inferior segment . Osteotomy lines were present 45 mm below the apices of the cuspids and 34 mm below the level of the mental foramina, so as not to jeopardize these structures. [710] care was taken to achieve sharp and straight cuts without creating curves . After mobilization of the lower segment, the chin was moved to the predicted position . The overlying soft tissue envelop was also inspected from the outside every now and then such that there is free mobility of the soft tissues surrounding the implant . A chin dressing with mild compression was placed after the operation to reduce swelling, to assist in mentalis closure, and to prevent hematoma or seroma formation . The patients were kept on a complete liquid diet for a week and progressed to a soft and then regular diet afterward . Sliding genioplasties were performed for seven female and one male adult patients presenting with a complaint of small or weak chins . The mean amount of horizontal chin movement was 10.75 mm . During the same period, eight chin augmentations with medpore the esthetic results, as determined by both the patient and surgeon, were satisfactory during the follow - up period of 3rd week to 6 months [figures 29]. In medpore group, there was no resorption of hard tissue and soft tissue decreased by a mean value of 2.82 mm over a period of 6 months, while in osseous genioplasty group, there occurred a mean hard tissue resorption of 1.33 mm and soft tissue decreased by a mean value of 3.30 mm over a period of 6 months [table 1 and 2]. Preoperative full face photograph of a patient of medpore chin augmentation group (group b) preoperative profile photograph of a patient of medpore chin augmentation group (group b) postoperative full face photograph of a patient of medpore chin augmentation group (group b) postoperative profile photograph of a patient of medpore chin augmentation group (group b) preoperative full face photograph of a patient of osseous augmentation group (group a) preoperative profile photograph of a patient of osseous augmentation group (group a) postoperative full face photograph of a patient of osseous augmentation group (group a) postoperative profile photograph of a patient of osseous augmentation group (group a) analysis of changes in hard and soft tissues (group a) (n = 8) analysis of changes in hard and soft tissues (group b) (n = 8) 0 in the medpore group, no infection or alloplastic reaction was found . The osseous genioplasty group experienced no infections, nonunion, or malunion . A mild and transient lower lip numbness developed in only one patient . The osseous genioplasty group experienced no infections, nonunion, or malunion . A mild and transient lower lip numbness developed in only one patient . It is a biologically inert polymer with regularly spaced pores measuring 100300 m in diameter . This pore size allows significant tissue ingrowth instead of surrounding fibrous tissue capsule formation, which results in rapid, strong implant fixation . It has been used as an onlay graft for augmentation of the cranium, mandibular angle, orbital rim, malar region, and mental region . The material is easily carved and somewhat flexible . Long - term follow - up evaluation demonstrated no problems related to bioincompatibility and a minimal complication rate (3% infection and 8% displeasing contours). Advancement genioplasty with horizontal osteotomy of lower border of mandible is a versatile, commonly carried out orthognathic procedure . Horizontal osteotomy of lower border of mandiblular symphysis through the intraoral approach was first introduced by richard trauner and hugo obwegeser . Horizontal osteotmy of chin can correct a horizontal or vertical deficiency or excess of chin . The procedure is suitable for any chin, short or long, and for laterogenia . In the medpore group, i.e. Group b, no infection or alloplastic reaction physically, medpore is a pure, biocompatible, strong substance that does not resorb or degenerate . It is stable in the long term with a good tensile strength, resistance to stress and fatigue, and a virtual lack of surrounding soft tissue reaction . Encapsulation and predisposition to movement are responsible for the majority of late complications reported for smooth surface implants (nonporous implant). Screw application of the implant to the underlying skeleton allows precise predictable contouring, thus limiting the need for revisional surgical procedure . When the hard tissue and soft tissue changes were compared, it was found that the hard tissue to soft tissue ratio was 1:0.87, 6 months postoperatively in group a, while in group b the hard tissue to soft tissue ratio was 1:0.78 . This shows that horizontal osteotomy of lower border of mandible, i.e., osseous genioplasty gives better soft tissue predictability than alloplastic augmentation. [1619] another aspect of this study has been to evaluate the ease of technique while using medpore biomaterial implants . Out of the 16 cases the average time of the surgery beginning from the placement of incision to closure of the site were noted . The average time of operation in the cases treated by medpore was 45 minutes to 1 hour, and for those treated by osseous genioplasty, it was 1.52 hours . Medpore produces the same satisfactory result as osseous genioplasty in cases of mild to moderate horizontal chin deficiency . All the patients from both the groups showed significant improvement in facial profile, high degree of satisfaction and the resulting improved self - esteem from both procedures.because of good fixation, medpore is quite different from traditional alloplastic implant . It did not depress into underlying bone, so the result was maintained, i.e., showed no resorption of underlying bone.surrounding tissue has shown biocompatibility to medpore biomaterial implant, which remains nonimmunogenic, nonallergic and nontoxic, with no evidence of resorption or alteration.more invasive nature of osteotomy in case of osseous genioplasty leads to the potential for more complications, more significant swelling and longer postoperative recovery.medpore implant can be given when only straight advancement is indicated and no vertical or asymmetric movement is indicated.in this advancement procedure, the soft tissue follows the skeletal framework of the chin . Hard tissue to soft tissue relation in case of osseous genioplasty was 1:0.88, while in the case of medpore augmentation it was 1:0.77, i.e., soft tissue predictability of osseous genioplasty was better in comparison with medpore augmentation . Medpore produces the same satisfactory result as osseous genioplasty in cases of mild to moderate horizontal chin deficiency . All the patients from both the groups showed significant improvement in facial profile, high degree of satisfaction and the resulting improved self - esteem from both procedures . Because of good fixation it did not depress into underlying bone, so the result was maintained, i.e., showed no resorption of underlying bone . Surrounding tissue has shown biocompatibility to medpore biomaterial implant, which remains nonimmunogenic, nonallergic and nontoxic, with no evidence of resorption or alteration . More invasive nature of osteotomy in case of osseous genioplasty leads to the potential for more complications, more significant swelling and longer postoperative recovery . Medpore implant can be given when only straight advancement is indicated and no vertical or asymmetric movement is indicated . In this advancement procedure, the soft tissue follows the skeletal framework of the chin . Hard tissue to soft tissue relation in case of osseous genioplasty was 1:0.88, while in the case of medpore augmentation it was 1:0.77, i.e., soft tissue predictability of osseous genioplasty was better in comparison with medpore augmentation . In brief, medpore is indicated for mild to moderate horizontal chin deficiency and to modify chin shape, i.e., for minor contour irregularity, while osseous genioplasty is recommended for any chin, i.e., short, long or for laterogenia.
In many countries particularly, small ruminants play a great role in the economy of the country, as sources of meat, milk, fiber, cash income, and skin and they can live in extreme climatic conditions, they can exploit herbage, which is unsuitable for large ruminants, and they require few labor - intensive inputs . Ethiopia lies within the tropical latitudes of africa and has an extremely diverse topography, wide range of climatic features, and multitude of agroecological zones, which make the country suitable for different agricultural production systems . This in turn has contributed to the existence of a large diversity of farm - animal genetic resources in the country . Ethiopian livestock production systems are broadly characterized as low input, mixed crop - livestock, agropastoral, and pastoral systems, as well as medium input, periurban, and urban enterprises . Ethiopia has the largest livestock and draft animal population in the african continent which is estimated to around 3040 million tropical livestock units (tlu) out of the total population in africa . There are approximately 49,297,898 cattle, 25,017,218 sheep, 21,884,222 goat, 7,582,625 equine, 759,696 camels and 38,127,504 chickens are found in the country . Generally, sheep are the predominant livestock in area of high lands of 3,500 meters above sea level; sheep assume a great share in socio - economic activities of about 85% of the population . Small holders in the high land area where is mixed crop - livestock population is practiced own most sheep in ethiopia, these sheep are an integral part of the livestock sector of the economy . Sheep supply meat, wool / hair and skin that generate about 89% of the farmer's cash income . Other special attributes of sheep over the other livestock resources include that they are highly adaptable to broad ranges of environment, have short generation cycles, and have high reproductive rates which lead to high production efficiency and poor people can afford few ewes since cost of them is less than a cow . With little inputs, sheep play an important role in the rural economy through provision of meat, milk, cash income, accumulating capital, fulfilling cultural obligations, manure and contribute to the national economy which can be incurred due to the export of live animals, meat, and skins . Despite the large livestock population of ethiopia, the economic benefits remain marginal due to prevailing diseases, poor nutrition, and poor animal production system, reproductive in efficiency, management constraints, and general lack of veterinary care . These diseases have a major impact on morbidity and mortality rates, with annual losses as high as 3050% of the total value of livestock products of ethiopia . Endoparasites are responsible for the death of one - third of calves, lambs, and kids and considerable losses of parts of carcasses condemned during meat inspection . It is well recognized that, in resource poor regions of the world, helminthes infections of sheep and goats are major factors responsible for economic losses through reduction in productivity and increased mortality . Nematode parasites of small ruminants result in low productivity due to stunted growth, poor weight gain, and poor feed utilization . In the tropics, the most important nematode species affecting small ruminants are haemonchus contortus, trichostrongylus species, cooperia species, bunostomum species, and oesophagostomum species, whereas nematodirus species are found in the high land areas of most of the country [9, 10]. The principal abomasal worms of sheep are haemonchus contortus, ostertagia circumcincta, ostertagia trifurcata, and trichostrongylus axei . Haemonchus contortus is one of the most important abomasal worms of sheep which is known as red stomach worm or wire worm of small ruminants . Haemonchus contortus is a species most commonly found in sheep and goat but haemonchus placei is the usual species in cattle and even so cross infection may occur when small ruminants and cattle graze together but the infestations are usually of less severity . Although helminthes parasites of ruminant livestock are ubiquitous in all of the agroclimatic zones of ethiopia with prevailing weather conditions that provide favorable condition for their survival and development, their presence does not mean that they cause overt disease . Among the diseases that constrain the survival and productivity of sheep, gastrointestinal nematode infection ranks highest on a global index with haemonchus contortus being of overwhelming importance . Haemonchus contortus found in abomasum of sheep and goat causes blood loss resulting in decrease in erythrocytes, lymphocytes, hemoglobin, packed cell volume, body weight, and wool growth . The abomasal nematode haemonchus contortus, which is particularly important and causes severe anemia and death in severely infected animals, identified haemonchosis as one of the top ten constrains to sheep and goat rearing in east africa . The first and second stages of larvae are free - living organisms and the host ingests the third stage larvae starting the infection . Adults of the parasite are found on the surface of the mucosa (the lining of the stomach). Both the larvae (l4) and the adults of haemonchus species suck blood . A thousand haemonchus species of adult can suck 50 ml of blood / day causing severe anemia . All ages of sheep are susceptible to haemonchus species infection but lambs are more susceptible than adults . A hematocrit reading of less than 15% is always accompanied by extreme weakness and shortness of breath and warrants a grave prognosis; less of plasma protein results in anasarca frequently manifested externally as a submaxillary edema (bottle jaw). The appetite typically remains good and, in acute outbreaks, affected animals may not lose appreciable weight . Feces are well formed, diarrhea occurring only in infections complicated by the presence of such species as trichostrongylus species and cooperia species . Lambs are the most seriously affected members of the flock, but older sheep under stress also may have fatal anemia . While, in temperate regions, the severity of gastrointestinal (gi) parasitic disease in most livestock farms is now minimized through the seasonal use of anthelmintics and pasture management, the problem persists in the vast majority of tropical and subtropical regions . Among the gastrointestinal parasites, it is important to assess the type and level of parasitism in ruminant livestock, in order to be able to determine the significance of parasite infection and to recommend the most beneficial and economically acceptable control measures . The determination of the risk factors associated with parasite occurrence can be used to design an effective control strategy [16, 17]. Previously there was not any documented data with regard to the prevalence of the parasite in the study area but there are high populations of sheep in the study area . Therefore, the study was designed with the objective of assessing the prevalence of ovine haemonchosis and its associated risk factors . This study was conducted in wukro which is located 848 km to the north of addis ababa and 45 km far from mekelle town in the eastern administrative zone of tigray region with an altitude of 134730n and longitude of 393557e . It is situated in area having an elevation of 1977 m above sea level . The area has clearly defined rainy season from july to september followed by long dry season from october to june . The study animals were 384 indigenous breed of sheep reared using traditional way slaughtered at various restaurants of wukro woreda . In this study the origins of the animals were recorded from the history of the animal owners . In addition, the age of the sheep was characterized using teeth eruption appendix a and body condition scoring method (medium and good) as per appendix b. for this study, the total population of sheep in the study area and expected prevalence of 50% was used using the 95% level of confidence . Hence based on the formula indicated by 384 sheep were examined: (1)n=1.962p expected1p expectedd295%=level of confidence, where n is sample size (384 animals), p is expected prevalence (50%), 1.96 is the value of z of 95% confidence level, and d is desired absolute precision = 5% . A cross - sectional study using random sampling was conducted from november 2013 to april 2014 to determine the prevalence of haemonchus species in sheep at the study site . The origin, age, sex, body condition, and general health condition of the animal was properly recorded . Then the abomasa were opened along their greater curvature and close visualization was made for the presence of adult haemonchus parasite . The worms were collected in normal saline . Then the parasites were identified based on the characteristics given by, appendix c. the data collected were entered to microsoft excel spread sheet, coded appropriately, and transferred in to spss version 20 software . For data analysis descriptive statistics was employed and statistical significance was considered when p value is less than 0.05 . In this study a total of 384 sheep were examined using postmortem for the presence or absence of haemonchus contortus and the result revealed that 157 (40.9%) were positive . Similarly, the prevalence of the parasite in sheep having different body condition indicated that there is significance difference of the parasite higher in medium body condition animals with the rate of 27.3% compared to good body condition sheep having the rate of 13.5% (table 1). Moreover, there was statistical significant variation in prevalence of haemonchus contortus among the studied age group with higher prevalence in young animals compared to adults in p <0.05 (table 2). At the same time there was statistically significant variation in prevalence of haemonchus in male and female with higher rate in male sheep compared to female (p <0.05) (table 3). The prevalence of the parasite in sheep that originated from different sites indicates that it was higher in those sheep originated from negash (25.3%) while the lowest was recorded in sheep originated from wukro (7.6%) but there was no significant variation (p> 0.05) as indicated in table 4 . The present study revealed that the overall prevalence of haemonchus contortus in sheep was 40.9%, which indicated high prevalence of the parasite in the study area . This finding was much lower compared with the study conducted by in gonder who reported prevalence of 80.21% . Similarly, the current finding is lowered compared to different researchers such as in wellega who recorded prevalence of 88.2%, with the rate of 93.6% in the ogaden region, and 96.5% in eastern part of ethiopia . Compared to the finding of who reported the prevalence of 37.18% in multan abattoir, the current finding was higher . With regard to the body condition of the examined sheep the rate was higher in medium body condition sheep compared to the good body condition sheep with the prevalence of 27.3% and 13.5%, respectively . This result is inconsistent with the previous reports of who reported prevalence of 77.21% and 84.44% in good and medium body condition animal, respectively . Similarly, indicated that the rate of the parasite was higher in medium body condition sheep compared to that of good body condition with the prevalence of 81.2% and 73.6%, respectively . The prevalence variation of the parasite in these two body conditions varies significantly (p <0.05). The current finding also indicated that there was significant difference of the prevalence of the parasite in young and adult sheep (p <0.05) where it was higher in young sheep compared to adult sheep with the rates of 21.9% and 19%, respectively, but indicated that the prevalence in young and adult sheep was 86.9% and 86.57%, respectively . Regarding the sex distribution of the parasite, the current finding revealed that the prevalence in male and female sheep was 29.7% and 11.2%, respectively . At the same time, reported that the rate was higher in male with the prevalence of 80.9% compared to female having the rate of 77% . In contrary to the current finding, reported that the rate of the parasite was higher in male compared to female with the prevalence of 34.11% and 39.22%, respectively . In support of the finding of indicated that the rate of the parasite was higher in female than male with the prevalence of 81.9% and 76.9% respectively . The prevalence of the parasites in sheep that originated from different sites of the study area also indicated that it was higher in those sheep originated from negash with the rate of 25.3% followed by agulae and wukro with the rates of 8.1% and 7.6%, respectively . However there is no statistical difference of the prevalence of the parasite (p> 0.05). The presence of high rate of haemonchus parasites in the study areas was responsible for the loss of production in sheep . The distribution of the parasites is more common in medium body condition and young animal, which needs great attention when designing the control programs of the parasite . This might be due to the practice of the society and ethical point of slaughtering of the male animals, as most of the female animals are kept for production purposes . Generally, the occurrence of the parasite among the sheep of the study site might be associated with the level of immunity of the animals as most of the young animals and those medium body conditions having low immune status are affected . In addition, the production level of the animals might contribute to the occurrence of the parasite as most of the sheep were using extensive grazing . Based on the current finding, the following points were recommended.the strategic deworming should focus on young and poor and medium body condition sheep.improvement of husbandry practices is very important.continuous surveillance of the parasite in sheep farm is essential.further study on the possible risk factors should be conducted.
Intrauterine adhesion (iua) is one of the important causes for infertility or recurrent pregnancy loss . In the past few years, iuas represented the significant clinical problem of women who mostly oppose infertility and there is no ideal therapy and thus it represents a major unmet medical need . Intrauterine adhesion results from trauma to increase the endometrial fibrosis . On the other hand iuas are bands of scar - link tissue that form between two surfaces inside the uterine cavity . It has been more than a century since heinrich fritsch first described a case of posttraumatic intrauterine adhesion . In general, adhesions are composed of fibrotic tissue, which may result in the adherence of opposing surfaces . It has been identified that the tissue injury is the specific risk factor and it is possible that, after an injury to the endometrium, fibrosis may follow with the potential for adhesion formation . Conversely, in the tissue the fibrin is rapidly deposited after trauma; then it either reverts or turns into fibrous adhesions . It varies still questionably in patients with experience of resorption or adhesion formation after trauma . In addition the characterizations of tissue fibrosis are considered to arise due to a failure of the normal wound healing response [3, 5]. During normal wound healing, a network of negative feedback mechanisms activated after successful healing is responsible for the proper termination of the proliferative and fibrotic processes, thus restoring tissue integrity . Deregulation of this healing process results in subsequent continuous secretion and deposition of ecm that may lead to the development of tissue fibrosis . In contrast, the repetitive trauma to the tissue may lead to continuous activation of fibrin proliferative response . However, the exact cause of deregulation healing process and continuous deposition of ecm components is still enigmatic . Conversely, the facts of clinical experience of fibrosis may consider as a somewhat permanent scar tissue, during which resolution of the healing process does not occur . Tgf-1 stimulates synthesis of ecm proteins and inhibits matrix degradation, resulting in the promotion of fibrosis and tissue repair . Tgf-1 belongs to a family of growth factors of critical importance in wound healing and fibrogenesis . Also, platelets are an abundant source of tgf-1; this isoform is released by degranulating platelets at the site of an injury, hence tgf-1 isoform consideration to play the essential role in wound healing and possible subsequent fibrosis . It has been studied extensively in models of liver, kidney, and lung fibrosis [79]. The tgf-1 is secreted as part of an inactive tripartite complex consisting of a homodimer of the tgf-1 with latency associated peptide (lap) and a molecule of latent tgf binding protein (ltbp). The complex is transported to the ecm and is the major form of tgf1 found in vivo [10, 11]. Nevertheless, tgf-1 is an important growth regulator of human endometrium; the various cellular responses elicited by tgf-1 are mediated through the activation of serine / threonine kinase receptors from the cell surface to the nucleus . Once activated, tgf-1 binds its receptors and then activates its downstream signaling mediators of smad3, to exert its biological activities [1316]. Altered expression of tgf-1 in human endometrium has also been associated with various abnormalities including extracellular matrix deposition and adhesion formation [1719]. Deregulation of the tgf-1/smads signaling pathway is responsible for tissue fibrosis in several organs . On the other hand, the involvement of smad3 is observed in fibrosis in many animal models of scleroderma, cystic fibrosis [20, 21]. Identified a number of collagen gene promoters in human dermal fibroblasts which were induced by tgf-1 and it depends upon smad3 . It has been recognized that the several abnormalities of endometrium occur with the action of tgf-1 which smads mediate [23, 24]. It is well accepted that smad3 is a critical downstream mediator responsible for the biological effects of tgf-1 . Alternatively, smad7 is an inhibitory smad and negatively regulates smad3 activation by its negative feedback mechanism . Expression of smad7 is induced by tgf-1, which, in turn, exerts its negative feedback mechanism by causing degradation of tri and smads; however, the overt expression of tgf-1 and smad3 and decreased smad7 are mostly observed in renal, cardiac, and bleomycin (bmi) induced lung fibrosis; this above evidence suggests that tgf-1 acts by stimulating smad3 to mediate fibrosis . Conversely, given the importance of tgf-1 in regulating several endometrial cellular activities, the expression and regulation of smads are necessary to mediate these actions . Besides, specific inhibitor of smad3 (sis3) to inhibit endothelial - myofibroblast transition and renal fibrosis in a type 1 diabetic kidney disease demonstrates a therapeutic potential for kidney disease by targeting smad3 signaling . On the other hand, sis3 is a specific inhibitor of smad3 phosphorylation via inhibiting the effects of tgf-1 . Furthermore, it has been reported that sis3 abolishes the ecm overexpression in the normal dermal fibroblasts and scleroderma fibroblasts in vitro . Based on the knowledge of the basic properties of tgf-1, we aimed to investigate tgf-1/smad3 signaling pathway and regulatory mechanisms of inhibitory smad7 may be essential in iua remodeling . Therefore, we demonstrated the expression of tgf-1, smad3, and smad7 in both iua patients and experimental rabbit model . Furthermore, we designed a study to evaluate the possible therapeutic effects of sis3 in experimental rabbit model . We carried out our study on 60 patients of iua, mean age 28.06 years (range 1934), taking a sample of peripheral venous blood and eleven samples of endometrial fibrosis tissue from march 2014 to december 2014, that were enrolled in this study and that were selected to be of moderate type of the iua . The score and the grade of the iua were evaluated during hysteroscopy examination according to the revised criteria (table 1) of the american fertility society (afs), finding where tissue samples from patients undergoing hysteroscopy adhesiolysis for infertility are . Exclusion criteria were infection, any disease in the uterus, chronic inflammatory and malignant disease, and any major operation within the previous 3 months . All patients had signed a written informed consent before surgery and had agreed to the collection of tissues for research . We carried out our study on 30 fertile women with mean age 28.43 years (range 1934) taking samples of peripheral venous blood and endometrial tissue biopsy of nonpathological site of uterine cavity to observe the expression of tgf-1, smad3, and smad7; inclusion criteria of control women included normal menstrual cycles, no history of previous reproductive surgery or chronic systemic disease, and no hormonal or immune modulator therapy for at least 6 months before the initial examination and being matched for age, having hormone reports within normal limit, and not being hospitalized for at least 1 month prior to participation . The study was approved by the local medical ethics committee of the third xiangya hospital, central south university, hunan, china . Experimental protocols were approved by the institutional animal ethics committee of the third xiangya hospital, central south university, hunan, china . From july 2, 2014, to july 23, 2014, 18 mature female fertile new zealand white rabbits weighing 25003000 g, age 58 months, were enrolled in this study . They had free access to food and water under standardized laboratory conditions in a temperature and light controlled environment for 2 weeks . Female rabbits weighing 2500 to 3000 g (age 5 - 6 months) were used in all experiments, and each underwent operation under aseptic conditions after induction of general anesthesia with pentobarbital sodium (30 mg / kg). Our aim was to propose a model of the pathogenesis condition in the rabbit similar to intrauterine adhesion observed in the human . Tissues were cut out and collection of inferior vena blood of rabbits 2 weeks after operation was performed . Seven rabbits were operated for a 0.51.0 cm longitudinal incision in the middle of the uterus, and the upper or lower half of the endometrium was randomly scraped using the sharp side of a uterine curette . All rabbits were sacrificed after operation each at 2 weeks after curettage and the abdominal incisions of the rabbits were sutured after flushing the uterine and peritoneal cavities . Tissues were cut out and collection of inferior vena blood of rabbits 2 weeks after operation was performed . After uterine curettage, performed in an identical manner to that of the endometrial curettage group, then 3 m sis3 (sis3 was used after diluting with solution in dmso) was intrauterine applied; the abdominal incisions of the rabbits were sutured in 2 layers with continuous 2 - 0 chromic catgut, after flushing the uterine and peritoneal cavities . Rabbits were killed for the collection of uterine tissue and inferior vena blood 2 weeks after operation . Two rabbits were operated and dmso solution in the uterus was injected for 2 weeks as an experimental control group . Tissues were cut out and collection of inferior vena blood 2 weeks after operation was performed . In the normal group (n = 2), were euthanized only for collection of endometrial tissue and blood samples which were obtained from the inferior vena cava . Peripheral 5 ml blood samples (human and rabbit) were obtained in k2-edta coated tubes and processed within 30 min of collection using two - step centrifugation . Samples were first centrifuged at 1.500 g for 15 at 4c . The supernatant was collected and then centrifuged again at 14.000 g for 15 at 4c to obtain pure plasma . Finally, plasma was transferred to rnase - free tubes and stored at 80c until rna extraction . Furthermore, tissues of both iuas and controls (human and animal) were snap frozen in liquid nitrogen and then stored at 80c until use . We measured transforming growth factor-1 (tgf-1) in plasma obtained from human and rabbit groups by an enzyme - linked immunosorbent assay (elisa) according to manufacturer's instructions . We used real - time quantitative reverse - transcription polymerase chain reaction (rt - qpcr) to detect the mrna expression of smad3 and smad7 in human and rabbit tissue . Total rna from different samples was isolated by using trizol reagent (takara, dalian, china) and the concentration and purity of rna were determined spectrophotometrically . Firstly, 4 l of pure rna (od 1.82.2) was reverse - transcribed (rt) to cdna at 42c for 30 minutes using reverse - transcription kits (takara, dalian, china) according to the instructions of the manufacturer, through an rt - pcr system (bio - rad, usa). Secondly, 2 l of cdna was used as the template in qrt - pcr . Then, smad3 and smad7 mrna expression were detected by using sybr premix ex taq (takara, dalian, china) according to the manufacturer's protocol, using a 7300 real - time pcr system (applied biosystems, ca, usa). Briefly, a 10 l reaction mixture containing 2 l cdna template, 5 l sybr master mix, 0.20 l rox, 2.4 l h2o, and 0.20 l of each primer was amplified by the following thermal parameters: initial incubation at 95c for 15 s, followed by 40 cycles of denaturation at 95c for 5 s, annealing, and extension at 60c for 31 s. the real - time pcr primers were shown in table 3 . Data analysis was performed by comparative ct method using the abi software . For the measurement of smad3 and smad7 mrna expression, -actin was served endogenous control and the results were expressed as the ratio of smad3, smad7 mrna to -actin mrna . Human and rabbit tissues were harvested and lysed with ice - cold protein lysis solution and protein lysates were centrifuged at 12,000 g for 15 min at 4c . Total protein in the supernatant was determined by a bca protein assay kit (beyotime, shanghai, china). Samples containing 3060 g of proteins were separated by 10% sds - page gel and transferred to polyvinylidene fluoride (pvdf) membranes . The membranes were blocked with 5% nonfat milk in tbst buffer (100 mm nacl, 10 mm tris - hcl, ph 7.4, and 0.1% tween-20) for 1 hr at room temperature . The membranes were then incubated with primary antibodies against smad3, p - smad3 (1: 200; santa cruz, ca, usa), smad7 (1: 500; santa cruz, ca, usa), tgf1 (1: 200; santa cruz, ca, usa), or -actin (1: 4000; santa cruz, ca, usa) with gentle agitation at 4c for 18 h, followed by horseradish peroxidase - conjugated secondary antibodies (goat anti - rabbit igg, 1: 1000; santa cruz; anti - mouse igg, 1: 6000; santa cruz) for 60 min at room temperature . The protein bands were determined by luminata crescendo western hrp substrate (millipore, billerica, ma, usa) through molecular imager chemidoc xrs system (bio - rad, philadelphia, pa, usa). Quantity one 1-d analysis software (national institutes of health, usa) was utilized for densitometry analysis . Finally, the protein expression results were measured as the ratio of target (smad3, p - smad3, smad7, or tgf1) density to endogenous control (-actin) density . Data were analyzed with spss software (version 20.0, spss, chicago, il) and reported as mean standard deviation (sd). Differences among groups were compared using student's t - test, one - way anova for categorical variables, fischer's exact test, or the chi - square () test and for non - gaussian distribution, rank transformation and wilcoxon test were used . The smads and tgf-1 expression data were used and graphs were made with graphpad prism version 6 for windows (graphpad software, san diego, ca, usa) and presented as mean standard error . All tests were 2-sided and a significant leveled p <0.05 was considered to be statically significant . A total 60 iua patients and 30 control women matched for age, history of normal menses, hypomenorrhea, cyclical lower abdominal pain, and abortion were enrolled in this study . The laboratory findings including rbc, wbc, hb, fsh, lh, prolactin, estrogen, progesterone, and testosterone hormones records were collected, respectively . There were no statistical differences between study group and control group (p> 0.05). We measured plasma concentration of tgf-1 in iua patients and intrauterine adhesion nontreated group of rabbit to determine whether tgf-1 was correlated with iua or not (figure 3). We found tgf-1 was significantly increased 2.7- and 7.1-fold in iua patients and intrauterine adhesion nontreated group of rabbit, compared to that in controls (p <0.05). Thus, increased concentration of tgf-1 is established as a key factor in iua and smad3 is proposed as a major player in tgf-1 signaling pathways that lead to iua . In this conception, further study to demonstrate the therapeutic effect of sis3 in the expression of tgf-1 in intrauterine adhesion and sis3 treated group of rabbit was performed and it was examined by using elisa . We found a marked 2.5-fold decrease of tgf-1 in intrauterine adhesion and sis3 treated group compared to intrauterine adhesion nontreated group of rabbit (p <0.05). Our results indicate sis3 exhibited a strong inhibitory effect of tgf-1 signaling in iua via the inhibition of smad3 . Also, smad3 is considered to be the primary signaling molecule involved in tgf-1 signal transduction in iua, because tgf-1 elicits potent profibrotic responses in iua and smad3, which are activated by tgf-1 and are indispensable for most of its profibrotic actions . Therefore, the inhibition of smad3 is a necessary component for repressing iua, which is inhibited tgf-1 that enhances reduction of the occurrence of the iua . Furthermore, we confirmed this result within tissue fibrosis in iua patients and experimental rabbit by rt - qpcr and western blot analysis . It has been evident that tgf-1 plays an essential role in fibrosis, which is regulated by smad3 . It is well known that the tgf-1/smad3 signaling system for an autoinhibitory loop involves smad7; as a result, the expression of smad7 influences the activity of tgf-1 . As an initial experiment, we measured the fibrous tissue expression of mrna and protein smad3, smad7, and tgf-1 in patients and experimental induced rabbits to determine whether tgf-1/smads differentially regulate their expression in iua or not . Consistent with the rt - qpcr (cdna marker), revealed that mrna expression of smad3 was significantly increased 2.8-fold, while mrna expression of smad7 was significantly decreased 2.1-fold in patients compared with controls (p <0.05) (figure 4). Further, our study demonstrated protein expression of tgf-1, smad3, and smad7 by western blot . Significant 3.8- and 2.5-fold increases of protein expression of tgf-1 and smad3 were noticed while protein expression of smad7 significantly decreased 5.01-fold in patients compared to controls (p <0.05) (figure 5). The mrna expression of smad3 was significantly increased 3.9-fold, while mrna expression of smad7 was significantly decreased 2.7-fold in experimental iua rabbit compared with controls (p <0.05) (figure 6). In this conception, we therefore examined the protein expression of p - smad3, smad3, smad7, and tgf-1, in iua rabbit (figure 7). Total protein expressions of smad3, p - smad3, and tgf-1 were markedly increased 2.6-, 3.2-, and 2.01-fold while protein expression of smad7 was significantly decreased 4.04-fold compared to that in controls (p <0.05). Similar results were obtained using fibrous tissue of experimental rabbit, in contrast to iua patient . However, these findings suggest that increased smad3 and decreased smad7 are an important mechanism underlying the action of tgf-1 activity in iua . Next, we investigated the possible effects of specific inhibitor of smad3 (sis3 with dose of 3 m) in iua which was associated with increased smad3, p - smad3, and tgf1 and decreased smad7 . It has been reported that sis3 with a dose of 3 m is able to reduce the smad3 and inhibited smad3 phosphorylation and tgf-1 induced fibrotic response in fibroblasts . Few studies have demonstrated that pretreatment of cells with sis3 (3 m) in human dermal fibroblasts was completely abrogated to tgf-1 signaling that contributes to the upregulation of type i procollagen via smad3 . Consistent with these findings, the present study demonstrates that the expression of fibrous tissue mrna smad3, smad7 and protein of smad3, p - smad3, smad7, and tgf-1 were determined by rt - qpcr and western blot analysis, respectively (figures 6 and 7). We found the mrna and protein expression of smad3 were markedly decreased 1.6- and 2.6-fold, while smad7 was significantly increased 2.4- and 3.1-fold in the intrauterine adhesion and sis3 treated group compared to intrauterine adhesion nontreated group (p <0.05). We also found the p - smad3 was decreased 3.1-fold in treated group compared to nontreated group of iua rabbit (p <0.05). Further, we found fibrous tissue protein expression of tgf-1 was significantly decreased 2.01-fold in the intrauterine adhesion and sis3 treated group compared to the intrauterine adhesion nontreated group (p <0.05). Together, these results indicated that sis3 inhibits the upregulation of smad3 by the suppression of the smad3 phosphorylation and increased smad7 was associated with inhibitory effect of tgf-1 in iua . However sis3, which inhibited increased tgf-1 signaling, could be used as a therapeutic intervention for iua via the inhibition of smad3 . This study described for the first time the role of individual expression of tgf-1, smad3, and smad7 signal pathway in iua patients and experimental rabbit model . We investigated in patients and created a rabbit model of postoperative iua using the endometrial curettage to establish a similar condition of iua in human, which will be help in our understanding of molecular mechanisms of intrauterine adhesion and also help to evaluate appropriate therapeutic alternative for prevention and treatment of the iua . In the past decades, extensive studies have been recognized in the role of various cytokines and growth factors that can lead to the development of tissue fibrosis in different organs . Among them, tgf-1 is a multifunctional regulatory cytokine with crucial roles in fibrotic processes . Therefore, it is important that only a few studies about the potential role of tgf-1 in fibrosis have been published so far [31, 32]. It is well accepted that active tgf-1 binds its receptors and then activates its downstream mediators, smad2 and smad3, to exert its biological effects, including fibrosis [33, 34]. The basis for tgf-1 regulation of collagen synthesis has developed rapidly as previous studies identified by smad family . However, smads component is a major specific signal transduction pathway of tgf1 . In the present study, we determine the expression of tgf-1, smad3, and smad7 levels in iua patients and experimental rabbit model to identify the molecular defects underlying the tgf-1 action . We divided three experimental approaches: in the first approach we demonstrated patient plasma for tgf-1 and fibrous tissue for mrna and protein that has expressed the regulatory smad3 and inhibitory smad7 in addition to tgf-1, respectively . In the second approach, the delay in the regeneration of endometrial epithelial cells may be important to the formation of iua after endometrial damage . Because of the insufficient epithelial cells covering the uterine cavity, the stroma is bared, allowing fibroblast activity and connective tissue formation leading to endometrial fibrosis and possibly to the development of the iua . With our experimental design the whole process of injury or trauma caused by sharp, deeply endometrial curettage could be evaluated and the partial clearance of endometrium was preceded that may contact the bare endometrium or myometrium and then caused more injury effect . Due to epithelial loss or epithelialization delay, the presence of fibrosis, or the varying degrees of scars, adhesions began to appear . Usually the epithelialization did not start until 2 weeks after injury; considerable granulation tissue, hyperplasia, and fibrosis appeared . When a sufficient percentage of fibrosis appears, it will hinder the regeneration of the endometrium and formation of the adhesion [36, 37]. However, trauma to endometrium goes through pathological changes and limited repair consistent with the clinical observation in iua patient . Our purpose was a model of the pathogenesis condition in the rabbit similar to iua observed in the patient . Indeed, our initial experiment of rabbits, the time points of 2 weeks after endometrial curettage has been characterized as the early stage of iua with healing scar may produce fibrosis overt an iua stage respectively, despite the fact that tgf-1 has powerful stimulation in tissue adhesions and continuous tgf-1 activation is associated with pathological cause of the iua . In the present study, confirms to establish iua a rabbit model by visible to the naked eye, plasma concentration of tgf-1 and tissue mrna and protein expression of smad3, smad7, and tgf-1 . In the third approach, the study was to assess the possible therapeutic effect as a treatment of intrauterine application of sis3 after endometrial curettage of iua rabbit that was compared with the intrauterine adhesion nontreated group of rabbit . Elisa analysis was used to determine the expressions of plasma tgf-1, rt - qpcr were used to determine smad3 and smad7 mrna, and western analysis was used to determine total smad3, p - smad3, smad7, and tgf-1 protein expression in both patient and rabbit model . In the present study, we demonstrated that the plasma concentration and protein expression of tgf-1 levels significantly increase in iua patients and experimental rabbit model compared to those in controls, respectively (p <0.05). Previously, data showed that increased tgf-1 induces fibroblasts to synthesize and contract ecm; this cytokine has long been believed to be a central mediator of the fibrotic response [10, 27]. On the basis of this conception our results indicate increased expression of tgf-1 levels have preceded the formation of fibrosis that enhances the development of the iua . Consistent with our observation, increased tgf-1 has been shown in atrial fibrosis, as well as transient overexpression of porcine tgf-1 in rat lungs using the adenovirus transfection system, induced prolonged lung fibrosis . Moreover, tgf-1 is expressed at greater levels in keloid fibroblasts when compared with normal dermal fibroblasts . There was an also growing evidence support that tgf-1 stimulates the progression of cardiac fibrosis during cardiac hypertrophy and heart failure . Indeed, in the present study, we found the marked different expression of tissue mrna and protein smad3, smad7 in patients and experimental rabbit model; we addressed this issue and found in other fibrotic disease; therefore our study believes these different expressions of smad may alter the development of the iua . However, the expression of smads in iua as well as their isolated adhesions tissue indicates that these adhesions possess the necessary components of the tgf-1 signaling pathway that can be recruited and activated by smads component . A significantly increased tgf-1 level had an effect on the expression of smad3 and smad7 in the iua . It has been evident that smad3 plays a role in the development of pathological fibrosis in different organs; for consistence in this regard, we further demonstrate the tissue mrna and protein expression of smad3 levels in iua . We found the tissue mrna and protein expression of smad3 levels are significantly increased in patients and experimental rabbit model compared to those in controls (p <0.05). We also measured phosphorylation of smad3 that significantly increased in the experimental iua rabbit model compared to controls (p <0.05). Our result suggests the altered tgf-1 expression has changed the smad3 expression and activation that may be more important in inducing the fibrosis in the iua . However, increased expression of smad3 is equally important as an increased expression of tgf-1 in the development of the iua . Also, tgf-1 is acting as a key regulator to change the smad3 expression in iua . Thus, previously published data taken together with our present study propose the existence of species specificity in the profibrotic effects of tgf-1 on endometrial stroma [17, 2528]. Consistent with our observation, few studies have shown increased smad3 expression in scleroderma skin, scleroderma fibroblasts, and postinfarction myocardial fibrosis [27, 28, 39]. Takagawa et al . Also demonstrated the expression of smad3 increased in a murine model of bm - induced scleroderma; these observations have initiated and facilitate our study to establish that smad3 is an important step in signal transduction or the activation of the iua . In the present study, we believed the iuas occur due to the abnormal responses or functional activities exhibited by tissue fibroblasts towards the mediators of tgf-1 which smads mediate . On the other hand, few studies demonstrated the evidence that found an underlying correlation between decreased smad7 and skin lesions, in a murine model of scleroderma and myocardial fibrosis in infracted heart [20, 21, 28]. It was previously demonstrated that the smad7 present within the cytoplasm of endometrium that regulates tgf-1 ligand initiated signaling by competing with the receptor regulated smads for receptor based phosphorylation and activation by an autocrine / paracrine action of tgf- . Generally smad7 not only is constitutively expressed, but also appears to be rapidly induced by tgf-1 in different fibroblast including dermal primary fibroblasts [20, 21]. On the basis of our data increased levels of smad7 are opposed to the fibrotic actions of tgf-1 in iua, whereas decreased levels of smad7 are more responsible for fibrotic effect in iua . Consistent with these findings, our result indicates the iua associated with marked decrease of tissue mrna and protein expression of smad7 and it will probably challenge the homeostatic role of smad7 in modulating of tgf-1 responses . As an inhibitory regulator in the tgf-/smad signaling pathway, smad7 can be induced by a smad3-dependent mechanism which in turn blocks the signal transduction of tgf-1 via its negative feedback loop [25, 4143]. Elevated tgf-1 level and decreased smad7 level are often present in tissues where an uncontrolled fibrotic response occurs . This finding is consistent with our present study in which we found the tissue mrna and protein expression of smad7 levels are significantly decreased in patients and experimental rabbit model compared to those in controls (p <0.05). Decreased smad7 is induced activation of smad3 via tgf-1 dependent pathways; as a result smad3-mediated fibrosis occurs in iua . Previous studies also reported reducing smad7 by activating the smad3-dependent smurf2 degradation pathway [41, 43]. Reduction of smad7 is caused by ubiquitin - dependent protein degradation that could be mediated, at least in part, by smurf2 proteins and that plays an important role in the fibrosis . However, collectively evidence indicated that dysregulation of smad3 and smad7 was established to be one of the major mechanisms in mediating the fibrotic response in iua . In this regard, dysregulation of smad3 and smad7 in iua may regulate through decreased smad3 and increased smad7 simultaneously, which seems to be an effective strategy for treatment of the iua . In our experimental rabbit protocol, we observe the expression of smad3 at 2 weeks overt the control; these observations suggest that the prolonged activation of smad3 is important for the initiation to activation of tgf-1 signals . However, our experimental rabbit data show that, during the development of iua, there is an increase in tgf-1 signaling as shown by increase of smad3 in fibrous tissue; thus this was attempted to activate tgf-1 signaling by fibrous tissue as evidenced by the decreased expression of the smad7 . The action of tgf-1 is effective in iua, possibly due to a higher tgf-1 receptor expression in iua, resulting in increased smad3 and decreased smad7 expression . Further, the present study revealed the therapeutic effects of sis3 in experimental rabbit model . Tgf-1 is a major stimulus for tissue fibrosis by enhancing the synthesis of collagens and other matrix components [45, 46]. Smad3 are necessary for the transcriptional activation of collagen genes [48, 49]. On the other hand, several studies have evaluated the fibrosis on the basis of the accumulation of collagen in tissue, due to an imbalance between production and deposition of ecm components including collagen type 1, and it promotes increased tgf-1 and smad3 while decreasing smad7 [26, 5052]. Tgf-1 signaling contributes to the upregulation of type i procollagen via smad3 in human dermal fibroblasts [53, 54]. Consistent with the above findings, the present study found overexpression of smad3 due to increased phosphorylated smad3 in uterine fibrous tissues . Besides, decreased smad7 expression resulted in sustained activation of tgf-1 and smad3 signaling and this trend may be due to a loss of the inhibitory effect of smad7 on smad3 activation which is closely associated with fibrosis in iua . Furthermore, sis3 has been shown to inhibit collagen synthesis by fibroblasts and fibrosis in several different experimental models . Sis3 may reduce the upregulated expression of type i collagen through the inhibition of phosphorylated smad3 in scleroderma fibroblasts . From the above evidence, the present study recommended that sis3 is responsible for inhibition of phosphorylated smad3 and type i collagen expression in iua . Moreover, 3 m sis3 completely diminished the constitutive phosphorylation of smad3 as well as the excessive type i procollagen expression in scleroderma fibroblasts . These findings supported our results that increased phosphorylation of smad3 is an important cause of excessive ecm deposition and sis3 is a selective inhibitor of collagen type i that reduced the occurrence of fibrosis in iua . This finding has showed, consistent with previous studies, treatment with sis3 in fibrosis of several different experimental models . Therefore, in this study, we had aimed to examine the efficacy of sis3 in the treatment of an iua rabbit model . Conversely, we demonstrate the intrauterine application of a specific inhibitor of smad3 (sis3) that alters the expression of smad3 and smad7 activity, which enhances altering of the transcriptional response of tgf-1 in iua . In the present study, we found the marked decrease of expression of tgf-1 and smad3 levels while smad7 level significantly increased in the intrauterine adhesion and sis3 treated group compared to intrauterine adhesion nontreated group (p <0.05). Also, sis3 is a smad3 inhibitor and blocks smad3 signaling directly by inhibiting smad3 phosphorylation . Moreover, sis3 also acts as a smad7 agonist and inhibits smad3 signaling by inducing smad7, which reduce the fibrosis of iua (figure 8). This finding was consistent with other study in which there was inhibition of smad3 with a combination of naringenin (ng) and asiatic acid (aa) (ng is a smad3 inhibitor and blocks smad3 signaling directly by inhibiting smad3 phosphorylation whereas aa functions as a smad7 agonist and inhibits smad3 signaling by inducing smad7). However, smad3 plays such a critical role in mediating the pathobiology of fibrotic disease, as demonstrated by the previously cited studies; inhibition of smad3 signaling could be a prime target for intervention in fibrotic conditions . Although increased tgf-1 was shown to induce the decreased expression of smad7, this induction was transient and therefore indicated the existence of a negative feedback loop within the pathway and sis3 has the ability to alter this pathway rather than enhance reduction of the occurrence of the iua . In this regard also, sis3 recognizes the inhibitory receptor of smad3 signal transduction pathways that result in interruption of tgf-1 action . Sis3 acts as an important negative feedback inhibitor of tgf-1 signaling by blocking the activation of smad3 and preventing their interaction with activated tgf-1 receptors; as a result disruption of smad3 confers resistance to the development of the iua . Together all data in the present study establish that significantly increased the expression of tgf-1, smad3 while marked decreased smad7 levels in intrauterine adhesion lead to, alteration of tgf-1 intracellular signaling and the underlying mechanism of tgf-1 action . As a substitute specific inhibitor of smad3 (sis3) that selectively targets individual signaling pathways downstream of the tgf-1 receptor to significantly decreased tgf-1, smad3 while increasing smad7 levels, therefore likely to be more successful to elicit to reduce the fibrosis occurrence in experimental iua rabbit model . These results suggest that tgf-1 mediates action through smads, and most likely signaling pathways result in intrauterine adhesion, growth, and its regression by specific inhibitors of smad3 (sis3). Our findings indicate that alterations in the expression of tgf-1 and smad3 components occur during the development of iua in both patient and experimental rabbit model . The patients and rabbit blood have shown significantly increased plasma concentration of tgf-1 is markedly correlated to the development of the iua, and iua is promoted to the increased tissue expression of tgf-1 and smad3 and decreased smad7 . Therefore, treatments targeting the modification of tgf-1, smad3, and smad7 signaling via suppressing smad3 with sis3 may provide a new therapeutic strategy for iua.
Morphologically, spores of microsporidia may be spherical, ovoid, rod - shaped, or crescent - shaped, although most are ovoid (2). These organisms are microscopically defined by the presence of a nucleated sporoplasm, a coiled polar tube and an anchoring disk . They lack several eukaryotic organelles such as mitochondria, golgi membranes, and eukaryotic ribosomes (3). Historically, microsporidial spores were first recognized as the causative agent of pbrine (pepper) disease, severely affecting the silk - worm industry in france and italy during the mid-17th century (4). Thenceforth microsporidiosis have affected honeybee, fish, and mink industries have been compromised by microsporidiosis (5). Since 1985, microsporidia have been incriminated as a causative agent of opportunistic infections associated with persistent diarrhea and weight loss in persons with aids (68). At first, microsporidia were microscopically considered as primitive protozoa, but in the 1990s, molecular and phylogenetic evidences revealed relationship of these organisms and fungi (9). More than 1300 species of microsporidia were identified and divided into about 150 genera (10, 11). Enterocytozoon bieneusi is considered the most prevalent enteric species infecting humans worldwide, followed by e. intestinalis (12). Vertical transmission of microsporidiosis from mother to offspring has been represented in animals (rodents, rabbits, carnivores, and non - human primates) (13, 14). The presence of microsporidia in the respiratory and intestinal tracts of infected individuals and the excretion of spores in urine and feces illustrate that horizontal transmission is possible through fecal oral transmission, oral oral transmission, inhalation of contaminated aerosols, and ingestion of contaminated food and water (8, 15). Risk factors associated with microsporidiosis that support horizontal transmission include homosexual practices, intravenous drug use, and exposure to water in swimming pools and hot tubs as well as occupational contact with water contaminated with microsporidial spores (16, 17). Those organisms are involved in drinking water contaminant candidate list of the us environmental protection agency (usepa) (18). E. bieneusi is found in a variety of mammals including macaques, dogs, cats, cattle, llamas, raccoons, muskrats, beavers, foxes, otters and pigs (1924). To our knowledge scarce data is available concerning the occurrence of intestinal microsporidiosis in domestic animals in egypt . Therefore, the aim of the present work was to investigate the prevalence and species of intestinal microsporidiosis among animals in giza, egypt . A total of 869 fecal samples were separately collected from animals in giza, egypt in 20122013 . Fecal samples were collected from 108 dogs canis lupus familiaris, 104 cats felis catus, 98 cattle bos taurus, 116 buffaloes, bubalus bubalis, 83 goats the collected samples were carried out to the laboratory at the same day of collection . Fecal samples were separately homogenized with clean spatula and then divided into two equal parts . One part of each fecal specimen was preserved in 10% formalin solution (merck) and used for microscopic detection of microsporidial spores . The second part of each fecal sample was kept at 20 c until used for dna extraction . Ethyl acetate concentration method was used to concentrate the spores of microsporidia in fecal samples (25). The obtained concentrated pellet was fixed on a clean glass slide using absolute methyl alcohol (merck) and stained with modified trichrome (mt) stain (26). Stained smears were microscopically examined with oil immersion lens for the presence of microsporidia spores . Stained microsporidia spores appeared reddish with clearly defined edges and a vacuole against a green background . The preserved part of each fecal sample was washed with phosphate buffer saline (pbs) and centrifuged at 2500 g for 5 min . The supernatant was decanted and the remaining pellet was washed again two times as mentioned before . Two hundred microliters of fecal suspension were extracted using the qiagen qiaamp dna stool mini kit (qiagen, valencia, ca) with a modified protocol . The temperature of the initial lysis step was increased to 95 c for 5 min, followed by incubation with proteinase k for 4 h in a water bath at 55 c, and then 100 l of ae buffer was used for elution . The elution step was repeated to increase the amount of dna yield by reapplying the original 100 l to the spin column . Pcr was performed using three different diagnostic primer pairs: i) generic microsporidia primer pair (pmp1 and pmp2) to confirm the presence of microsporidia (27); ii) species specific primer pair (ebief1/ebier1) for amplification of microsporidial small subunit rrna (ssu - rrna) coding regions of e. bieneusi (28); and iii) species specific primer pair (sintf / sintr) for e. intestinalis (29) (table 1). Primers of microsporidia, enterocytozoon bieneusi and encephalitozoon intestinalis amplification of dna was performed using maxima hot start green pcr master mix (thermo scientific). A hot - start procedure for microsporidia and e. bieneusi was used with an initial denaturation at 95 c for 4 min followed by 35 cycles of denaturation at 94 c for 30s, primer annealing at 60 c for 30 s and extension at 72 c for 30s . A final extension step was performed at 72 c for 10 min (27, 28). The optimal pcr conditions for the sintf / sintr primers began with an initial denaturation at 95 c for 5 min, followed by 35 cycles of denaturation at 94 c for 30s, annealing at 55 c for 30s, and extension at 72 c for 90 s. a final extension step was performed at 72 c for 10 min (29). A total of 869 fecal samples were separately collected from animals in giza, egypt in 20122013 . Fecal samples were collected from 108 dogs canis lupus familiaris, 104 cats felis catus, 98 cattle bos taurus, 116 buffaloes, bubalus bubalis, 83 goats the collected samples were carried out to the laboratory at the same day of collection . Fecal samples were separately homogenized with clean spatula and then divided into two equal parts . One part of each fecal specimen was preserved in 10% formalin solution (merck) and used for microscopic detection of microsporidial spores . The second part of each fecal sample was kept at 20 c until used for dna extraction . Ethyl acetate concentration method was used to concentrate the spores of microsporidia in fecal samples (25). The obtained concentrated pellet was fixed on a clean glass slide using absolute methyl alcohol (merck) and stained with modified trichrome (mt) stain (26). Stained smears were microscopically examined with oil immersion lens for the presence of microsporidia spores . Stained microsporidia spores appeared reddish with clearly defined edges and a vacuole against a green background . The preserved part of each fecal sample was washed with phosphate buffer saline (pbs) and centrifuged at 2500 g for 5 min . The supernatant was decanted and the remaining pellet was washed again two times as mentioned before . Two hundred microliters of fecal suspension were extracted using the qiagen qiaamp dna stool mini kit (qiagen, valencia, ca) with a modified protocol . The temperature of the initial lysis step was increased to 95 c for 5 min, followed by incubation with proteinase k for 4 h in a water bath at 55 c, and then 100 l of ae buffer was used for elution . The elution step was repeated to increase the amount of dna yield by reapplying the original 100 l to the spin column . Pcr was performed using three different diagnostic primer pairs: i) generic microsporidia primer pair (pmp1 and pmp2) to confirm the presence of microsporidia (27); ii) species specific primer pair (ebief1/ebier1) for amplification of microsporidial small subunit rrna (ssu - rrna) coding regions of e. bieneusi (28); and iii) species specific primer pair (sintf / sintr) for e. intestinalis (29) (table 1). Primers of microsporidia, enterocytozoon bieneusi and encephalitozoon intestinalis amplification of dna was performed using maxima hot start green pcr master mix (thermo scientific). A hot - start procedure for microsporidia and e. bieneusi was used with an initial denaturation at 95 c for 4 min followed by 35 cycles of denaturation at 94 c for 30s, primer annealing at 60 c for 30 s and extension at 72 c for 30s . A final extension step was performed at 72 c for 10 min (27, 28). The optimal pcr conditions for the sintf / sintr primers began with an initial denaturation at 95 c for 5 min, followed by 35 cycles of denaturation at 94 c for 30s, annealing at 55 c for 30s, and extension at 72 c for 90 s. a final extension step was performed at 72 c for 10 min (29). Microscopic examination of 869 fecal samples from different animals revealed the presence of intestinal microsporidia in 17.0% of them by using modified trichrome stain . The highest rate of infection with intestinal microsporidia was recorded in dogs (33.3%), followed by 23.1, 20.5, 18.8, 14.9, 14.3, 11.2, 9.1 and 6.9% in cats, goats, pigs, rabbits, cattle, sheep, donkeys and buffaloes, respectively (table 2 and fig . 1). Prevalence of intestinal microsporidia in animal fecal samples by modified trichrome stain original picture of different microsporidial spores stained with mt stain . Bar = 5m molecularly, intestinal microsporidial spores were recorded in 95 (64.2%) out of the 148 microscopically positive samples . 76.8% and 42.1% of the pcr positive animal fecal samples had e. bieneusi and e. intestinalis, respectively . The highest rate of infection with e. bieneusi reached 100% in cattle, buffaloes, sheep and goats . The highest rate of infection with e. intestinalis was recorded in donkeys (100%), followed by 82.6, 36.4, 31.3, 26.7, 25.0 and 20.0%, in dogs, goats, cats, pigs, buffaloes and rabbits, respectively . Dual infection with both e. bieneusi and e. intestinalis was recorded in examined fecal samples from buffalo, rabbit, goat, cat, dog and pig (table 3 and fig 24). Species identification of intestinal microsporidia in examined animals by pcr ethidium bromide stained 2% agrose showing pcr products of microsporidia . M: marker (100 plus bp), ve: negative control, samples 1,2,3: positive samples at 250279bp . M: marker (100 plus bp); ve: negative control; lanes 13: positive samples ethidium bromide stained 2% agrose showing pcr products of enterocytozoon bieneusi . M: marker (100 plus bp); ve: negative control; lanes 13: positive samples overall, 148 out of 869 examined animal fecal samples were positive for intestinal microsporidia by mt stain . Only 95 out of 148 microscopically positive fecal samples were molecularly positive for intestinal microsporidia . This result agreed with another study (30) where the detection of microsporidia by pcr showed lower correlation with their detection by light microscopy . Furthermore, in portugal the inhibitors could interfere with pcr results and therefore pcr was postulated to have a limited value for identification of the species of microsporidia present in fecal samples (31). In the present study, a total of 14 (14.3%) cattle fecal samples were positive for intestinal microsporidia by modified trichrome stain . Other workers in spain found no infection with intestinal microsporidia in fecal samples from three caws by weber s chromotrope stain and this might be attributed to the small number of samples (32). In mexico, e. intestinalis was detected in one caw fecal sample stained with quick - hot gram chromotrope stain (33). The present result showed that nine out of 14 microscopically - positive cattle fecal samples were molecularly - positive and identified as e. bieneusi . In other molecular studies, e. bieneusi was reported in cattle feces in germany (11.7%) (34), usa (9.5%) (35), korea (14.9%) (36) and argentina (14.3%) (37). In our study, a total of 8/88 (9.1%) donkeys had intestinal microsporidia by mt stain . (32) where one of two examined fecal samples of donkeys had intestinal microsporidia and bornaylinares et al . (33) who examined only two donkeys and found that one of them had intestinal microsporidia . The greater difference in the number of examined samples could not be dependent due the very low number of examined animals . The present results showed that 6 out of 8 donkey fecal samples (positive for microsporidia by mt stain) had intestinal microsporidia by pcr . In usa, molecular examination of only 2 donkeys revealed that one of them had intestinal microsporidia identified as e. intestinalis (33), while in spain no microsporidia was detected by pcr in donkey fecal samples that were microscopically positive for microsporidia (32). Microscopic examination of pig fecal samples stained with mt stain in the current investigation revealed the presence of microporidial spores in 18.8% of them . A higher occurrence (82%) of intestinal microsporidia in pigs, no intestinal microsporidia was observed in 4 pigs by using weber s chromotrope stain and this might be attributed to the low number of the examined fecal samples (32). The current work revealed that 15 out of 18 pig fecal samples (that were microscopically positive for microsporidia) were positive by pcr . The identified species of microsporidia in these samples were: 12 pigs had e. bieneusi, four pigs had e. intestinalis . Other workers recorded higher incidences (94% and 92.6%) of e. bieneusi by pcr in the examined pig fecal samples from czech republic and slovakia (38, 39), while lower incidences were reported in switzerland, massachusetts (usa) and japan (4043). To our knowledge there were no available data concerning the detection of intestinal microsporidia in sheep by using staining techniques . Using pcr technique in the present work, it was found that 60% of microscopically positive sheep fecal samples for intestinal microsporidia were pcr positive and all samples had only a single infection with e. bieneusi . Other workers agreed with our result in that e. bieneusi was the only species identified in feces of sheep (43). In the present work, 20.5% of the examined goat fecal samples (n=83) by mt stain were microscopically positive for intestinal microsporidia . Other workers in spain observed clusters of microsporidia - like spores within a vacuole inside epithelial cells in fecal smears of one goat using weber s chromotrope - based stain (32). In the present study, in addition, 41.2% of pcr positive goat fecal samples had a single infection with e. bieneusi . Other workers in spain (32) and peru (43) found that 14.2% and 2% of the examined goat fecal samples had e. bieneusi only, while bornay - linares et al . (33) in usa examined one goat fecal sample by pcr and identified e. intestinalis in it . 14.9% of the examined rabbit fecal samples had intestinal microsporidia by mt stain which was higher than the result of lores et al . (32) in spain who found that 2/22 (9.1%) of rabbits had intestinal microsporidia by weber s chromotrope stain . Molecularly, single as well as mixed infections with e. bieneusi and e. intestinalis were observed in pellets of rabbits in the present work . E. bieneusi was microscopically identified and by pcr for the first time in rabbit fecal samples in spain, a single infection of rabbits with either e. bieneusi (44) or e. cuniculi (32) was recorded, while no infection with intestinal microsporidia was reported in germany (34). Intestinal microsporidia were microscopically detected in cat feces in our study in egypt (23.1%) and in portugal (29.4%) (31), but not in spain (32) might be attributed to difference in geographic criteria of these different countries . The present study showed that both e. bieneusi and e. intestinalis were present in feces of cats . In germany (34), japan (42) and usa (45), e. bieneusi was the only identified species in 5, 17 and 14.3% of cat fecal samples by pcr, respectively . In portugal, both e. bieneusi and e. cuniculi were detected in feces of cats (31), while no intestinal microsporidia were found in ten examined cat fecal samples in spain (32). In the present study, 36/108 (33.3%) of dogs had intestinal microsoporidia by mt stain . Other workers in spain found a lower incidence (5.9%) of intestinal microsporidia in dogs by weber s chromotrope stain (32). In portugal, a lower incidence [13.8% (5/36)] of intestinal microsporidia was detected in dogs by mt stain (31). The variation between our result and the previous results (31, 32) might be attributed to difference in the number of the examined samples . The present work declared that dogs could have e. bieneusi, e. intestinalis or both in their feces . Other workers in colombia (46), spain ((32), portugal (31) and japan (42) found molecularly that 15, 11.8, 60 and 2.5% of examined dogs had intestinal microsporidia belonging to only e. bieneusi, while in no microsporidial spores were found in 60 dog fecal samples examined by pcr in germany (34). E. bieneusi was the most prevalent intestinal microsporidia in domesticated animals that may play a role in dissemination of intestinal microsporidiosis in the environment.
The innate immune system provides a first line of defense against various microorganisms and is essential for the control of pathogenic infections . The adaptive immune system has evolved to provide a more versatile means of defense which prepares increased protection against subsequent reinfection with pathogen . Although the innate immune system cannot always recognize or eliminate infectious pathogens, play a crucial part in the initiation and subsequent direction of adaptive immune responses, as well as participating in the removal of pathogens that have been targeted by an adaptive immune response . Moreover, because there is delay before the initial adaptive immune response takes effect, the innate immune response has a critical role in controlling infections during this period . The innate immune system is mediated by pattern recognition receptors (prrs) that primitive part of the immune system through pathogen - associated molecular patterns (pamps) (magnadttir, 2006). Lectins belong to the prr class and a group of sugar binding proteins that recognize the exposed carbohydrates on the cell surface of potential pathogenic microbes, and then agglutinate various cells by binding to cell - surface glycoconjugates (saraiva et al ., lectins act as a mediator of self and non - self - recognition and play a significant role in cellular functions, including cell communication, agglutination, proliferation, opsonization, phagocytosis, signal transduction, metastasis and apoptosis (wassaman et al ., 1986; sharon & lis, 2004). A fish lily - type lectin sequence was identified in snakehead murrel (channa striata), large yellow croaker (larimichthys crocea), spotnape ponyfish (leiognathus nuchalis), bartail flathead (platycephalus indicus), turbot (scophthalmus maximus), rock bream (oplegnathus fasciatus), orange - spotted grouper (epinephelus coioides) (arasu et al ., 2013; tasumi et al ., several articles were published that lectins are involved in the immune response against pathogen infection (choi et al ., 2015; kim et al ., 2011; park et al ., 2012; thulasitha et al ., however, these studies are limited to information that the lectin is involved in the innate immune response . The rock bream (oplegnathus fasciatus) is a commercially important marine fish and it has a high economic value in the south korea . Although the recent rapid development of the rock bream farming, the spread of bacterial and viral diseases causing enormous economic loss of the aquaculture industry . Rock bream iridovirus (rbiv) is one of the major pathogens in rock bream aquaculture and identified as the most important pathogen infecting rock bream in the last decade (inouye et al . However, there is little available information based on temporally integrated observations in the immune responses against virus infection . Therefore, research on immunity - related genes in rock bream is necessary to cope with widespread disease infection . In the previous, ofltl-1 has been named as rbltl and reported that changes of expression in the ontogeny, spatial and pathogen infection response (park et al ., 2016). Although the cdna sequence of the ofltl-2 and -3 has been reported, no relevant study on the in vivo function of ofltls has been conducted . In this study, we investigated that domain analysis and sequence alignment, expression changes in the developmental stage, tissue - specific distribution and expression profile in the immediate and long - lasting immune response against rbiv infection . The amino acids are numbered along the right margin as well the top of the sequences . The genbank accession numbers of lily type lectins are gu565039 (ofltl-2) and gu565040 (ofltl-3), respectively . The cdna and amino acid sequences of ofltls were analyzed using the basic local alignment search tool (blast) at the national center for biotechnology information website (http://blast.ncbi.nlm.nih.gov/blast.cgi) and the expert protein analysis system (http://www.expasy.org), as reported previously (wilkins et al ., 1999). The brood stock of rock bream (5 years old) were reared in the genetics and breeding research center of the national institute of fisheries science (nifs, geoje, republic of korea) with aerated seawater and natural photoperiod in a 30-ton tank . When the water temperature was 23.01.0, photoperiod maintained on a 15h light: 9h dark to induce the maturation of gonads . After spawned naturally, the fertilized eggs in the same clutch were collected by a blotting silk net and incubated with weak aeration and fresh seawater at 23.50.5. after 25 h, the larvae hatched and were placed in a 15-ton tank at a density of 46 larvae / l . Larvae were fed with the l - type rotifer brachionus plicatilis from 3 to 20 days after hatching (dah) and artemia nauplii from 16 to 30 dah, and then gradually switched to extruded pellet food beginning at 25 dah . During the rearing time, the rock bream larvae and juveniles were maintained in the conditions as below: water temperature (24.01.0) and salinity (32.50.5%). Experimental analysis for tissue distribution including brain, eye, gill, intestine, kidney, liver, skin, spleen and stomach samples were dissected from ten healthy rock breams (total length of approximately 10 cm, 5 - 6 months old) and immediately frozen in liquid nitrogen, followed by storage in a -80 freezer until use . The rock breams were anaesthetized prior to experiments involving tissue collection and pathogen injection, and samples were collected under aseptic conditions . All fish were acclimatized to experimental condition for 1 week before processing . For the rbiv challenge experiment, the rock breams were divided randomly into two groups: a control group and a challenged group . The control and challenged fish were injected with 100 l of phosphate buffered saline (pbs) or a rbiv suspension (10 tcid50 virus/ fish), respectively (umasuthan et al ., 2013). The temperature to which the experimental fish were subjected was controlled at 20 using a re - circulation system, without flow and feeding . Challenged fish collected under aseptic conditions at 0, 3, 6, 12, 24 and 72 hours post - injection and pooled together in equal amounts and frozen in liquid nitrogen . Total rna was extracted from the ground fish with tri solution (bsk - bio co.) as described in the manufacturer's protocol . The total rna was treated with dnase - i (sigmaaldrich) to remove genomic dna contamination . The concentration of total rna was quantified using spectrophotometrically (biotek, gen 5.2) and rna quality was assessed via electrophoresis in 1% agarose gels . Cdna was synthesized with suprimescript rt premix (2x) (gennet bio) using an oligo (dt) primer . To synthesize cdna, the reverse transcription reaction was conducted as follows: a mixture containing 1 g of total rna, the oligo (dt) primer and rnase - free dh2o was held at 65 for 5 min, then placed on ice for 5 min . Collect the contents of the tube by brief centrifugation and add 10 l 2x suprimescript rt premix, and then incubates at 60 min at 50. the final inactivation reaction was carried out for and 10 min at 70. specific primers for rock bream lectins and -actin were designed using the primer 3 program (table 1). Expression analysis of rock breams lectins was conducted using quantitative real - time pcr with specific primers, and the mrna levels of -actin were used as an internal control . Quantitative real - time pcr was conducted using the abi 7500 real - time detection system (applied biosystems) according to the manufacturer s instructions . The final reaction volume contained 10 l of fast sybr green pcr master mix (applied biosystems), 100 ng of cdna, 0.3 l of each of the forward and reverse primers . The amplification procedure consisted of an initial denaturation step for 20 seconds at 95, then 40 cycles of 3 seconds at 95 and 30 seconds at 60, followed by a final dissociation stage . Dissociation curve analysis of the amplification products was performed after quantitative real - time pcr to confirm the specificity of the pcr products . The relative expression ratio of the target gene versus the -actin gene was calculated using the 2ct method (pfaffl, 2001). All samples were analyzed with three duplicates, and all data are presented in terms of relative mrna levels, expressed as the meanse (n=3). Statistical analyses were performed with spss 17.0 software (spss inc .) And the data were subjected to one - way analysis of variance (anova). Differences were considered significant at p<0.05 () and extremely significant at p<0.01 ( ). Ofltls amino acid sequence do not have a signal peptide nor a trans - membrane region, but contains bulb - type mannose binding lectin(b - lectin) domain between 3 and 112 aa (total of 110 aa sequence) that typical features for its fundamental structure . A long dimerization interface site is also available within the b - lectin domain profile between aa 4 and 112 (total of 109 aa). The deduced amino acid sequence of rock bream lily type lectin-2 shared 69.2% identity with ofltl-3 . Ofltls two qxdxnxvxy motifs were completely conserved, but third d - mannose binding domain lack in the rock bream . To determine whether a role of rock bream lily - type lectins in the early developmental stage, temporal expression analysis was conducted using the samples from immediately after hatching to 60 days larvae and juveniles rock bream fish . For a quantitative analysis, quantitative real - time pcr was carried out using gene - specific primers designed from the ofltl-2 and 3 coding sequence . Expression of ofltl-3 was maintained the basal level without substantially increased until 9 dah, and it began to increase from 10 dah and rapidly was increased up to 60 dah (fig . The overall temporal expression pattern of ofltl-2 is similar to ofltl-3, although tran sient expression decreased in the early stage that is different with ofltl-3 (fig . Ofltls are not to expression from the early development, is an increased expression after immune - related organs is fully formed . To investigate the expression pattern of ofltls according to the developmental stage, using the sample from fertilization up to 60 days larvae and juveniles rock bream fish . After removing seawater from the sample, soaked in trizol solution and homogenized to extract total rna . (c) the development scheme of the immune system and expression profile of ofltls in the rock bream early developmental stages . Light gray bar shows the bottleneck when many larvae die (12 to 16 dah). To investigate the role of the rock bream lectins in vivo, relative mrna expression of each tissue was calculated using rock bream -actin as a reference gene and the result was further compared with the lowest expression tissue to determine the relative tissue - specific expression profile (set as 1). Ofltl2 and 3 were predominantly expressed in the liver and skin that the highest expression in the skin and the following order was liver . Also, the expression of ltls was almost at basal level except the liver and skin tissue, although the difference between the high - order depending on the gene (fig . The spatial distribution of (a) ofltl-2, (b) ofltl-3 in various tissues of healthy rock bream fish . The transcript level was determined via quantitative realtime pcr, and rock bream -actin was chosen as an internal reference gene . An asterisk indicates a statistically significant difference * p<0.01) compared with lowest expression the tissue (set as 1, black box). The results are reported as the mean standard deviation (sd) of triplicates . Significance was analyzed via one - way analysis of variance (anova) using the spss 17.0 program . To investigate the function of ofltls in the immune response to a viral infection, the temporal patterns of ofltls mrna expression were examined using the whole body of rock bream fish infected with rbiv . Expression patterns and timing are preferentially confirmed with rbiv - induced marker genes to verify a successful challenge experiment . The mrna level of cathepsin h expression was high in both the early (3h) and later stages (24h); these results are consistent with previous findings (kim et al ., 2013). 4, although expression of ofltl-2 was weak in the early response period, it gradually increased until 24 hours post - infection and then dramatically increased to 72 hours . While, expression of ofltl-3 maintaining almost the basal levels until 12 hours post - infection and it was exponentially in infected 24 hours post - infection, and then increased expression of ofltl-3 was reduced to 1/9 at 72 hours after infection . The ex - pression patterns of osltls were determined in the whole body (a) ofltl-2 and (b) ofltl-3 through quantitative real - time pcr . Cathepsin h was used as markers to ensure a successful rsiv infection expe - riment (inset in fig . The samples were analyzed at 0, 3, 6, 12, 24 and 72 hours post - injection . The expression of -actin was used as internal control for quantitative real - time pcr, and each experiment was performed in triplicate . An asterisk indicates a statistically significant difference (* p<0.05; * * p<0.01) compared with 0 h (set as 1). Significance was analyzed via one - way analysis of variance (anova) using the spss 17.0 program . In contrast to higher vertebrates, fish is living in aquatic environments from early embryonic stages of life, which are an ideal medium for microorganism growth compared to air and every moment is constantly exposed to a pathogen attack . The bony fishes are derived from one of the earliest divergent vertebrate lineages to have both innate and acquired immune systems . However, the adaptive immune response did not clearly reveal in fish and it is expected not sophisticated like mammals . Therefore, the innate immune system is the only defense weapons of invertebrates and a fundamental defense mechanism of fish and more dependent their innate immune systems for survival and the resistance to the pathogens . Lectins play important roles in the recognition and elimination of pathogens via the innate immune system . Researches on the immunological functions of lectin are concentrated in the innate immune response of higher vertebrates . However, our results seem to be involved both in the innate and adaptive immune response; lily - type lectins are expressed after completing immune system during early developmental stage and preferentially increased in the second half reaction in a viral challenge, these results are related to the adaptive immune response . On the other side, ofltls are expressed in the innate immune response related tissue . Because fish have not sophisticated and specialized the adaptive immune systems, the innate immunity - related genes will be able to engage in the long - lasting immune responses to infection . Research findings are not infrequently has been reported that the early and late results are quite cross . Therefore, outside the framework defined in the higher vertebrates various studies will be needed . In the development of the immune system rock bream, spleen anlage was observed between the swim bladder and the intestine at 5 dah, and the thymus was formed as a paired structure under the pharyngeal epithelium above the gill arch at 10 dah . The order of the immune organs becoming lymphoid was the pronephric kidney (10 dah), thymus (15 dah) and spleen (21 dah) (zhizhong et al ., 2013). Ofltls were hardly expressed in the initial developmental phase and increased from 10 dah . Thereafter, expression of ofltls was proportionate to development of rock bream immune organ . It can be inferred that they will participate in the immune response after the immune system is fully established, not involved from the initial development period . Fish are permanently exposed to various external hazards that aerobic and anaerobic bacteria, viruses, parasites, marine pollutants . To protect fish from pathogenic microorganisms, mucus acts as a physical, chemical and biological barrier between the fish and the environment which first site of interaction with pathogens . The mucus composition is very complex and includes immuneglobulins, agglutinins, lectins, lysins and lysozymes that are anti - microbial factor and is secreted from the fish skin . These factors are very important recognize the microorganisms invasion and to protect the fish from invading pathogens (lee et al ., 2015; suzukiet al ., 2003 the liver is unique anatomical and immunological organ: the typical function of the liver is carbohydrate, protein and lipid metabolism, bile secretion, antioxidation and detoxification . As well as, the liver play a critical roles against invading pathogen in the innate immune responses of fish that particularly enriched macrophages and lymphocytes compared with other organs and modulated of liver injury and recruitment of circulating lymphocytes (racanelli & rehermann, 2006; castro et al ., 2014). Although it does not clearly separated innate and acquired immune response dedicated organ in fish, liver and skin are primarily involved in many innate immune response . In this study, these results are to be expected that ofltls contribute to the innate immune response against viral infection, but the transcripts were increased to more intensively in the period of acquired immune of rbiv challenge experiments . For this reason, the further expression analysis is needed using a virus - infected liver and skin tissue of the fish because it uses the rsv infection whole fish in this experiment.
The world health organization estimates the number of anemic people worldwide to be very high (2 billion) and that approximately 50% of all anemia can be attributed to iron deficiency . Currently, the global prevalence of anemia is estimated to be 30.2% in nonpregnant women rising to 41.8% during pregnancy . Anemia prevalence among pregnant women is around 24.1% in the americas, 48.2% in south east asia, 25.1% in europe, 44.2% in eastern mediterranean, 30.7% in the western pacific, and the highest in africa at 57.1% [24]. In ethiopia, higher proportion of pregnant women are anemic (22%) than women who are breastfeeding (19%). Though anemia has multifaceted causes, half of its burden is attributed to iron deficiency (i d). According to the world health organization (who), 12.8% and 3.7% of maternal mortality in asia and africa, respectively, are directly attributable to anemia . The prevalence of anemia in pregnancy has remained unacceptably high worldwide, especially in developing countries over the past three decades . The world health organization has recommended a 6-month regimen of a daily supplement containing 60 mg of elemental iron along with 400 g of folic acid for all pregnant mothers . In areas with a higher prevalence of anemia accordingly, in ethiopia, the national guideline for control and prevention of micronutrient deficiencies highlights the need of daily iron supplementation for at least 6 months during pregnancy and 3 months postpartum . Despite this program, in ethiopia, <1% took an iron supplement for the recommended period (90 days or more) during their last pregnancy . Therefore, the major problem with iron - folate supplementation in pregnancy is compliance, as women often fail to take the supplements regularly as supplemented by their health workers due to varying factors [9, 10] and this is thought to be a potential driver to the persistent high prevalence of anemia in pregnant mothers . This emphasizes the need to study the factors influencing compliance with iron - folate supplements . Objective of the study general objectives: to assess compliance with iron - folate supplement and associated factors among antenatal care attendant mothers in misha district, south ethiopia, 2015 . Specific objectives: to determine status of compliance with iron - folate supplement among anc attendant mothers.to identify factors affecting compliance with iron - folate supplement among anc attendant mothers . To assess compliance with iron - folate supplement and associated factors among antenatal care attendant mothers in misha district, south ethiopia, 2015 . To assess compliance with iron - folate supplement and associated factors among antenatal care attendant mothers in misha district, south ethiopia, 2015 . To determine status of compliance with iron - folate supplement among anc attendant mothers.to identify factors affecting compliance with iron - folate supplement among anc attendant mothers . To determine status of compliance with iron - folate supplement among anc attendant mothers . To identify factors affecting compliance with iron - folate supplement among anc attendant mothers . A community based cross - sectional study using quantitative and qualitative data collection methods was conducted from march 1 to march 10, 2015, among randomly selected pregnant mothers in misha district . There are seven health centers and thirty - five health posts, which provide routine antenatal care services to the community . The study population consisted of sampled pregnant mothers in the study area who were attending anc and being supplied with iron - folate supplement . Pregnant mothers who did not participate in quantitative study and health extension workers were participants of qualitative study . Pregnant mothers who had stayed in antenatal care for at least one month preceding the study period and had received iron - folate supplementation were included in the current study . However, those who were unable to respond or very sick were excluded . The sample size was determined based on the single population proportion formula using z2 p q / d with a 95% ci, 5% margin of error, and an assumption that 74.9% of pregnant women are compliant with iron - folate supplement in the study area . Assuming a 10% nonresponse rate, a total sample size of 303 pregnant women was required . A total of 13 participants, including 8 pregnant mothers who were not involved in quantitative study and 5 health extension workers, participated in in - depth interview . Prior to the actual data collection, the list of study subjects was identified by using community health management information system (chmis) folder . Finally, the study participants were selected by using random numbers generated by a computer program . The criterion purposive sampling technique was used to select pregnant mothers and health extension workers for qualitative study the questionnaire was designed in english and translated into hadiyisa (a local language). In order to ensure that the original meanings of the items on the questionnaire were maintained, the hadiyisa version was translated back into english by a researcher conversant in both languages . The final hadiyisa version was pretested on a sample of 15 pregnant mothers attending anc and using ifa supplement in neighbor district or outside the study area . The questionnaire consisted of items that assessed sociodemographic characteristics, compliance to supplements, knowledge of anemia and iron - folate supplement, pregnancy and related experiences, and facility and supplement related factors . The outcome for this study, compliance to ifa supplement, was assessed based on the reported number of ifa tablets taken in the previous 7 days before the survey . Pregnant mothers who took at least 70% of the expected dose of the iron - folate tablets in the previous week before the study, which is equivalent to consuming at least five tablets per week, were considered as compliant with iron - folate supplement . The independent variables include respondent's sociodemographic characteristics (age, educational status, occupation, and income), pregnancy and related experience (gravidity, parity, and frequency of anc visits), health care related factors (distance to the nearest health facility, counseling about ifa), and knowledge of anemia and ifa supplement . Mother age was categorized into three groups by five - year interval, which was later recoded into two categories as <25 and 25 because they were very few cases per cell to run logistic regressions . The respondents' comprehensive knowledge of anemia was computed by summing up 18 multiple - choice items (4 items on the sign and symptoms of anemia; 6 items for anemia causes; 6 items for consequences of anemia during pregnancy; and 2 items on prevention and treatment of anemia during pregnancy). Comprehensive knowledge of iron - folate supplement was measured by summing up 8 multiple - choice items . A correct answer was given one mark, while a wrong answer was not given any mark . Data were entered into and cleaned using epi data version 3.1 and then exported to spss version 16 for further analysis . Then, binary logistic regression was used to examine the relationship between the proposed predictors and compliance to iron - folate supplement . Variables with p value 0.25 in the bivariate analysis were entered into multivariable logistic regression to identify variables independently associated with iron - folate supplement compliance . 95% ci with a respective odd ratio was used to assess the statistical significance of association among the variables . P value less than 0.05 was used as cut - off point to see the presence of statistically significant association . In - depth interviews were audio - taped and verbatim - transcribed into hadiyisa and then directly translated into english . Ethical clearance and approval for the study was obtained from the ethical committee of jimma university, college of health science . Thus, oral consent was obtained from all the respondents after explaining of the purpose of the study, risk / discomfort, benefits to the subject, confidentiality of records, right to refuse participation and terminate participation in the study at any time . From a total of 303 pregnant mothers, 296 were involved in the study, yielding a response rate of 97.6% . The mean age of the study participants was 29.07 (sd 6.0) years . 46.3% had a primary level of education followed by secondary and above (39.2%). The majority of the mothers' occupation was housewife (88.20%) (table 1). One hundred eighty (60.8%) respondents took <70% of expected doses of the ifa supplements, that is, for less than five days in a week, and 116 (39.2%) respondents took greater than or equal to 70% of expected doses of the ifa supplement in a week, that is, for greater than or equal to five days in a week . Amongst women who missed the doses of ifa supplement, the leading underlying reason was side effects (125, 50.6%). Of those respondents who reported missing of ifa doses due to side effects, 96 (76.8%) respondents reported heart burn, 19 (15.2%) respondents reported nausea and vomiting, and 10 (8%) respondents reported stomach cramping . The other reasons of skipping doses of ifa supplement were forgetfulness (104, 42.1%), perceived shortage of iron - folate supplements in health facility (13, 5.3%), and not liking the taste of ifa supplement (5, 2%). Finding from qualitative part of the study revealed that most pregnant mothers' main reason of missing dose of iron - folate supplement was fear of side effects of iron - folate tablet.i feel nausea and heartburn as soon as i took the tablets . [pregnant mothers, age 32] i feel nausea and heartburn as soon as i took the tablets . [pregnant mothers, age 32] another qualitative finding revealed that the second main reason forwarded by the in - depth interview participants was forgetfulness.i want to take the iron - folate tablet as prescribed, but i forget to take the tablet regularly . I want to take the iron - folate tablet as prescribed, but i forget to take the tablet regularly . [pregnant mothers, age 26] from the key informant interview, most of the participants argued that the main reason for missing the dose of iron - folate supplement was gastrointestinal side effect of the iron - folate tablets heartburn.some pregnant mothers reported the missing of doses of iron - folate tablets during their anc visits the reason they told me that they felt the heartburn (gastric problem) when they took the tablets . Some pregnant mothers reported the missing of doses of iron - folate tablets during their anc visits the reason they told me that they felt the heartburn (gastric problem) when they took the tablets . Comprehensive knowledge of anemia was computed from summing up all relevant 18 knowledge items (4 items on sign and symptoms of anemia; 6 items for anemia causes; 6 items for consequences of anemia during pregnancy; and 2 items on prevention and treatment of anemia during pregnancy). A correct answer for each item was scored as 1 and incorrect answer was scored as 0 . Items were then summed up and converted to 100% . Accordingly, the median score was 61.1, mode was 72.2, and the mean was 61.9 (sd = 17.4). About 56.1% of the respondents scored above the median value; therefore, they had good knowledge of anemia and the remaining scored below the median value . Comprehensive knowledge of iron - folate supplement was computed by summing up all relevant 8 items (3 items on benefits of iron - folate supplementation and 5 items on possible effects of iron deficiency anemia during pregnancy). A correct answer for each item was scored as 1 and incorrect answer was scored as 0 . Accordingly, the median score was 62.5, mode was 75, and the mean was 63.8 (sd = 21.9). About 61.5% of the respondents scored above the median value and the remaining scored below the median value . From total respondents, 261 (88.2%) of mothers were multigravida and 35 (11.8%) were primigravida . A high percentage of the pregnant mothers interviewed were in their third trimester (239, 80.7%) whereas 57 (19.3%) were in their second trimester . Concerning the parity of the respondents, 191 (64.5%) of mothers were multipara and 66 (22.3%) and 39 (13.2%) of respondents were primipara and nullpara, respectively . Of all respondents, 55 (18.6%) had a history of anemia during pregnancy confirmed by clinical health workers . Concerning the utilization of antenatal care, 261 (88.2%) of respondents visited anc for less than four times and 35 (11.8%) of respondents visited anc for greater than or equal to four times . As part of supplement related variable, only 5 (1.6%) respondents reported the disliking of taste of iron - folate supplement . Of the total respondents, 232 (78.4%) said that it took them 30 minutes or less to reach the nearest health institution from their residence, 62 (20.9%) of pregnant mothers said that it took 3060 minutes to reach the institution, and 2 (0.7%) mothers reported it took greater than 60 minutes to reach the nearest health institution . One hundred fifty - four (52%) of respondents got counseling on ifa tablets and 48% of respondents did not get counseling on ifa tablets . To investigate the association of predictors with ifa supplement, predictors which showed an association with ifa supplement at p value of less than or equal to 0.25 in the univariate were selected as candidate variables for multivariable logistic regression analysis . Among the variables entered to multivariable logistic regression, age of mothers, counseling on iron - folate supplement, knowledge of ifa supplement, knowledge of anemia, and frequency of anc visits pregnant mothers whose ages were 25 years 2.9 times more likely complied with iron - folate supplement than those pregnant mothers who were <25 years old (aor = 2.985, 95% ci = (1.069,8.340)). Pregnant mothers who had good knowledge of iron - folate supplement were 3.5 times more likely to be compliant with iron - folate supplement as compared to those who had poor knowledge about iron - folate supplement (aor = 3.509, 95% ci = (1.442,8.537)). Pregnant mothers who had good knowledge of anemia were 4.4 times more likely compliant with iron - folate supplementation as compared to those who had poor knowledge (aor = 4.451, 95% ci = (2.027,9.777)). Similarly, mothers who had visited anc four times and above were 3.5 times more likely compliant with ifa supplement as compared to mothers who visited anc less than four times (aor = 3.558, 95% ci = (1.189,10.653)). Pregnant mothers who were counseled on iron - folate supplement during pregnancy were 4 times more likely compliant than those who were not counseled on intake of ifa supplement (aor = 4.093, 95% ci = (2.002, 8.368)) (table 2). The result revealed that 39.2% of pregnant mothers were compliant (took at least 70% of the expected dose of the iron - folate tablets in seven days of the previous week of the study) to the supplement, which is much lower compared with the study done in four regions of ethiopia which was 74.9% and higher than study done in amhara regions of ethiopia that was 20.4% . The probable reason may be the difference in geographic locations and the time gap between studies and study subjects . Even though the compliance rate is low compared with other countries this could be due to differences in level of study (national and district level) and the time gap between the present study and edhs 2011 . The compliance rate among pregnant mothers in this study was still lower compared to studies done in other countries like united state, philippines, nigeria, and senegal [10, 1315]. This difference may be due to differences in awareness of pregnant mothers about iron - folate supplementation and study design . The side effect is repeatedly considered as a major problem with compliance . According to studies conducted in the philippines, senegal, and india, it was reported as a reason for missing doses of iron - folate supplementation among pregnant mothers by 20.2%, 27.0%, and 27.6%, respectively . Studies conducted in vientiane also concluded likewise . In this study, it was observed that, from those mothers who missed the doses of ifa supplement, 50.6% were because of fear of side effects . Other studies done in the amhara region of ethiopia and eight districts of four regions of ethiopia revealed that 54.4% and 63.3% of respondents, respectively, missed the dose of iron - folate supplement due to fear of side effects [11, 12]. One probable rationalization for the high figure of side effects in this study can be lack of information about potential side effects of iron - folate supplement in advance . Appropriate counseling is known to increase the psychological tolerance of pregnant mothers to side effects of iron - folate supplement . In this study, 42.1% of pregnant mothers skipped doses of iron - folate supplement because of forgetfulness and this finding is parallel with the findings in india and vientiane by 48.8% and 47.9%, respectively [13, 17], but it is much higher than the finding in ethiopia that was 16.7% . The discrepancy may be due to inadequate counseling of mothers to remember to take their tablet in current study . This problem could be addressed through better counseling during the anc visit by suggesting to women strategies to remember to take their tablets, for example, placing the tablets in a spot that they see every day . Fear of having big fetus because of consuming iron - folate tablet was obstacle to compliance with iron - folate supplement in previous study which was done in thailand . One study in ethiopia also showed that 28.45% of women believed that continuous taking of iron - folate supplementation leads to overweight babies . But, in this study, none of the respondents reported stopping of taking iron - folate supplement because of fear of having big fetus / baby . This could be due to differences in setting of study subjects . After adjusting for other factors in the regression analysis, one factor shown to have a significant association with women who were 25 years old were 2.9 times more likely to be compliant to iron - folate supplementation than women with younger ages (<25 years). The reason for this is that older women may be more concerned about their health and pregnancy outcomes and had better experiences in the prevention and treatment of iron deficiency anemia . This finding was in line with a study in india that elderly and middle women were slightly more compliant than younger women and also this finding is consistent with the study done in ethiopia . Similarly, visiting anc four and more times were considered to have significant effects on compliance with iron - folate supplement . Mothers who had visited anc for four and more times are 3.5 times more likely to be compliant with iron - folate supplement compared to mothers who visited anc for less than four times . The possible reason of this is that health providers may help mothers during their anc visits by discussing compliance with iron - folate supplement, encouraging them to take the tablet as prescribed, and educating them on health benefit of taking ifa supplement, and these help mothers to be compliant with iron - folate supplement . In this study, pregnant mothers who had good knowledge of anemia were 4.4 times more likely to be compliant with iron - folate supplement during pregnancy compared to those who had poor knowledge . This finding was consistent in nigeria, vientiane, and amhara region of ethiopia [10, 12, 17]. Pregnant mothers who had good knowledge of iron - folate supplement during pregnancy were 3.5 times more likely to be compliant with iron - folate supplement during pregnancy compared to those who had poor knowledge about iron - folate supplement . Similar finding was found in nigeria and amhara region of ethiopia [10, 12]. The reason could be that knowledge helps women to have a good perception of prevention and treatment of anemia during pregnancy by taking iron - folate supplement during pregnancy . Pregnant mothers who were counseled on iron - folate supplement during pregnancy 4 times more likely complied than those who were not counseled on ifa supplement . This finding is consistent with the study done in india (haryana state), sweden, cambodia, and senegal [9, 15, 20, 21]. Reports of the pregnant mothers may under / overestimate compliance rate since the data was collected by self - report . The compliance rate of iron - folate supplementation during pregnancy remains very low in the misha district . This indicates that the who and fmoh recommendations were no met even though there was usefulness of iron - folate supplementation program during pregnancy to prevent iron deficiency and iron deficiency anemia during pregnancy . In this study, ages of the mothers, counseling on iron - folate supplement, knowledge of anemia, knowledge of iron - folate supplement, and frequency of anc visits were found to be significantly associated factors of compliance with iron - folate supplementation during pregnancy . Furthermore, fear of side effects of iron - folate supplement, forgetfulness, and perceived shortage of iron - folate supplement in the health facility were commonly mentioned reasons for missing the doses of iron - folate supplement . Therefore, compliance with iron - folate supplementation can be increased by providing women with clear instructions about iron - folate tablet intake and educating them on the anemia and health benefits of the iron - folate tablets . Also, promoting mothers to visit anc at least four times can improve their status of compliance with iron - folate supplementation . Recommendation is made based on finding as follows: providing pregnant mothers with clear instructions about iron - folate tablet intake, educating them on anemia during pregnancy and its consequences and health benefits of the iron - folate tablets, and promoting mothers to visit anc at least four times . Also, further research is recommended on compliance with iron - folate supplement using the pill count method to overcome the limitation of this study.
One of the most important roles of feet is weight - bearing, which requires stability and resilience for absorbing impact, and the medial longitudinal arch (mla) and transverse arch provide this stability and resilience1 . In particular, the mla is the primary structure absorbing the impact of the weight - bearing that occurs during the physical activities of walking or running2 . The condition in which the mla becomes chronically or abnormally low is called flatfoot3, and it is divided into spastic flatfoot and flexible flatfoot4, 5 . The reported causes of flexible flatfoot include excessive extension of the plantar muscle, which provides stability to the mla, and poor function of the tibialis posterior muscle4 . Some researchers have reported that hip external rotator weakening internally rotates the hip joint while inducing foot pronation6 . Hip external rotator strengthening exercises, performed by extending the hip joint when it is in external rotation and the knee joint is at 90 flexion, have been reported to have the greatest effect when performed in the prone position7 . The results of one study showed that as the activity of the abductor halluces (abdh) increased, the angle of the mla decreased, indicating that abdh exercises are helpful for the maintenance and enhancement of the mla8 . In a study of various exercises for strengthening the abdh, toe - spread (ts) exercises and short - foot exercises performed in the sitting position did not elicit any significant difference in abdh activity, but the muscle activity value was shown to be higher during ts exercises9 . Although many studies have been conducted of the various exercises for mla correction10, 11, studies examining exercises that also strengthen the hip external rotator muscle are insufficient . Therefore, the purpose of the present study was to investigate the effect of hip external rotator strengthening and ts exercises in subjects with pronated feet, and to evaluate their effects on lower extremity muscle activities during stair - walking . A total of 20 healthy adults were selected after excluding those with present or past arthritis, those who had undergone foot or ankle surgery, and those with diabetes, cuts on the lower extremities, or foot abnormalities . The subjects were selected from among those with foot pronation (> 10 mm) based on navicular drop (nd) tests9, 12 . The participants were provided with a written informed consent form in accordance with the ethical principles of the declaration of helsinki . The mean age, height, and weight of the study subjects who performed both hip external rotator and ts exercises were 21.81.62 years, 162.96.9 cm, and 59.314.6 kg respectively, and the mean age, height, and weight of the study subjects who performed only ts exercises were 22.31.95 years, 165.08.1 cm, and 63.211.8 kg, respectively . A surface emg dts system (telemyo dts, noraxon inc ., az, usa) was used to record and process the electromyographic (emg) signals from the gluteus maximus, the vastus medialis, the tibialis anterior, and the abdh in order to measure the muscle activities of these muscles . The collected surface emg signals were processed using the myoresearch master 1.07 xp program (noraxon inc . The signals were digitally sampled at a rate of 1,500 hz and noise was removed using a 20500 hz bandpass filter and a 60 hz notch filter . The emg data were normalized using the maximal voluntary contraction (% mvc) values . The study subjects performed exercises five times per week for four weeks: two times per week at home and three times per week in the laboratory . The subjects who performed both hip external rotator exercises and ts exercises performed three sets of 20 repetitions of hip external rotator exercises in the prone position along with 100 repetitions of ts exercises in the sitting position while maintaining both the hip joints and the knee joints at 90 flexion . Nd tests12, 13 were conducted before and after the four weeks of exercises and the mean values of the groups were compared using the independent t - test . Before stair - climbing and -descending, foot switches were attached to the center of the heel, below the first metatarsal, and below the fifth metatarsal to divide the phases of weight bearing in order to measure the muscle activity of the gluteus maximus, the vastus medialis, the tibialis anterior, and the abdh during the individual phases . The paired t - test was used to analyze changes in lower extremity emg during stair - walking before and after the exercise period within each exercise group . The results were statistically analyzed using spss version 18.0 (spss inc ., chicago, il, usa) and significance was accepted for values of p<0.05 . The nd of the group that performed both hip external rotator and ts exercises was 5.661.58 mm and that of the group that performed only ts exercises was 7.522.16 mm after the intervention . These results indicate that the nd of the group that performed both hip external rotator and ts exercises decreased significantly more than the group that performed only ts exercises (table 1table 1.comparison of navicular drop test results between the group that performed both hip external rotator exercises and ts exercises and the group that performed only ts exercises (n=20)hip external rotator exercise and ts exercisemean (sd)ts exercisemean (sd)prend (mm)11.8 (1.9)11.4 (1.4)4 weeksnd (mm)5.7 (1.6)7.5 (2.2)nd: navicular drop, sd: standard deviation . P<0.05). According to emg measurements taken during stair - walking before and after the intervention, the% mvc values of the vastus medialis (5.567.27%) and the abdh (11.0510.54%) during stair - climbing and the% mvc values of the abdh (7.96.23%) during stair - descending were significantly different in the group that performed both hip external rotator and ts exercises (table 2table 2.emg activities:% mvc values pre- and post - intervention (n=10)musclehip external rotator exercises and ts exercisests exercises onlypremean (sd)4 weeksmean (sd)premean (sd)4 weeksmean (sd)stair upgm22.1 (7.8)24.4 (9)22 (9.1)25.8 (10.4)vmo31.1 (12.5)36.7 (12.9)45.5 (17)48.7 (5.7)ta21 (12.8)19 (11.3)17.2 (5.5)15.7 (6.1)abdh27.1 (4.7)38.18 (8.8)23.9 (7.3)37.3 (11.5)stair downgm13.6 (9)12 (5.1)15.4 (14.2)22.3 (12.8)vmo35.8 (21.7)32.5 (8.9)37.1 (15.5)41.2 (9.5)ta20.1 (10.7)19.8 (16)17.2 (10.2)20 (9.1)abdh32 (6.8)39.9 (9.6)25 (11.1)36.9 (9.7)gm: gluteus maximus, vmo: vastus medialis, ta: tibialis anterior, abdh: abductor hallucis, sd: standard deviation . The% mvc values of the abdh during stair - climbing (11.5813.46%) and stair - descending (11.8710.78%) were significantly different between before and after the exercise treatment (table 2). After the 4-week intervention, there was no significance different between hip external rotator and ts exercise and only ts exercise (table 3table 3.comparison of emg activities (% mvc) between the group that performed both hip external rotator and ts exercises and the group that performed only ts exercises after the 4-week intervention (n=20)musclehip external rotator exercises and ts exercisesmean (sd)ts exercises onlymean (sd)stair upgm24.4 (9)25.8 (10.4)vmo36.7 (12.9)48.7 (5.7)ta19 (11.3)15.7 (6.1)abdh38.2 (8.8)37.3 (11.5)stair downgm12 (5.1)22.3 (12.8)vmo32.5 (8.9)41.2 (9.5)ta19.8 (16)20 (9.1)abdh39.9 (9.6)36.9 (9.7)gm: gluteus maximus, vmo: vastus medialis, ta: tibialis anterior, abdh: abductor hallucis, sd: standard deviation). P<0.05 gm: gluteus maximus, vmo: vastus medialis, ta: tibialis anterior, abdh: abductor hallucis, sd: standard deviation . P<0.05 gm: gluteus maximus, vmo: vastus medialis, ta: tibialis anterior, abdh: abductor hallucis, sd: standard deviation the present study was conducted to compare changes in lower extremity muscle activities during stair - walking between subjects with flatfoot who performed only ts exercises and subjects who additionally performed hip external rotator exercises, in order to present basic evidence for exercise treatment . Since many studies set the criterion for flatfoot as 10 mm or greater nd12,13,14, the present study s subjects were selected based on that criterion . In the case of flatfoot, valgus stress is created in the knee joints during gait due to the pronation and extroversion of the hind foot, the supination of the fore foot and the mid foot, and the internal rotation of the lower extremities, leading to kinematic sway of the lower extremities and compensatory actions1 . Under the assumption that femoral anteversion contributes to hip joint internal rotation, some researchers have reported that the muscle activity of the vastus medialis and the hip adductor are decreased by hip internal rotation and 40 knee flexion15 . Based on these previous study results, in the present study, hip external rotator muscle strengthening is considered to have increased hip joint external rotation, leading to increased muscle activity of the vastus medialis during stair - climbing . The decrease in nd height that occurred after hip external rotator strengthening exercises would be explained by the internal rotation of the lower extremity, as described by neumann1 . Limitations of the present study include the fact that gait perse is an activity with large variations among individuals, and stair - walking also has limitations for generalization because individuals stair - walking characteristics are different . In addition, muscle activity levels and joint angles at different time - points could not be identified during motion, as the changes in muscle activity and the joint angles were evaluated together . The issues discussed here should be addressed in future studies to present evidence for exercise treatment through kinetic and kinematic analyses.
Alongside antithrombin and c1-esterase inhibitor, activated protein c (apc) represents an important coagulatory inhibitor in humans . The zymogen protein c is activated by the thrombin thrombomodulin complex to give apc, whose anticoagulatory activity is conferred primarily by its ability to inactivate factor va and viiia . Disruption of the normal balance between coagulation and fibrinolysis has been characterized by its correlation with organ dysfunction and increased mortality . Thus, acquired protein c deficiency is associated with increased morbidity and mortality in patients with severe sepsis and septic shock . Direct anti - inflammatory properties of apc that have been characterized in vitro were considered independent from coagulatory inhibition [7 - 9]. Thus, an experimental apc application is capable of blocking the lethal effects of escherichia coli - induced septic disseminated intravascular coagulation, but also improves outcome in meningococcal - induced septic shock . Conversely, the application of antiprotein - c antibodies produces higher sensitivity towards defined endotoxin stimuli and increases mortality . Because organ dysfunction and animal survival are not improved by the administration of other substances with comparable anticoagulant effects, it has been suggested that the action of apc is not solely dependent upon its anticoagulatory properties . Although there is in vitro evidence from cell - culture studies to show that apc blocks leukocytic adhesion to the endothelium, the in vivo mechanisms of apc - related anti - inflammatory and microcirculatory action have not been completely elucidated thus far . Before discussing the role of apc on the microcirculation in sepsis, we will provide some information on the role of the microcirculation in the development of organ dysfunction and coagulatory failure . Leukocyte endothelial - cell interaction is essential for an effective defense against bacterial invasion . However, leukocytic activation is associated with an increased proinflammatory immune response, which is caused by an increased release of inflammatory cytokines, including tumor necrosis factor-, interleukin-1 and interleukin-8, which initiate generalized neutrophil (polymorphonucleocyte) activation . This release of mediators, together with the generalized activation and sequestration of neutrophils, may contribute to the widespread microvascular injury and subsequent endothelial damage observed in sepsis . The migration of polymorphonucleocytes to inflamed tissue is primarily dependent on the induction of secondary adhesion molecules that are expressed on leukocytes and activated vascular endothelium . This migration, along with the subsequent release of oxygen radicals and other toxic substances, represents a central step in the development of multiple organ failure . The endothelial switch from anticoagulant to procoagulant during sepsis is thought to be related to the cytokine - mediated expression of endothelial adhesion molecules (e.g. E - selectin, p - selectin, intercellular adhesion molecule-1) and increased tissue factor production by monocytes and endothelial cells . Additionally, endotoxins are, per se, known to directly generate an environment favoring coagulation processes by activating the extrinsic coagulation pathway via monocytic tissue factor expression . Thus, the concept of exogenous application of natural coagulatory inhibitors has been evaluated in terms of reducing systemic activation of leukocytes and endothelial cells . Although the anticoagulatory effects of apc have been extensively investigated during the last two decades, only a few studies have analyzed the actions of apc on the microcirculation during ischemia reperfusion . To study ischemia reperfusion injury in a clinically relevant setting, models of warm ischemia have been extensively used in rodents . In a rat model interestingly, the histological changes in renal tissue observed in control animals after noncrushing microvascular clamping for 60 min were almost completely absent after prophylactic apc administration in this model . These apc effects on organ structure corresponded to a highly effective preservation of renal tissue blood flow that was significantly decreased after ischemia reperfusion in controls . Since these postischemic alterations were only significantly inhibited by apc and not a specific factor xa inhibitor or heparin, the authors suggested that apc protects against ischemia reperfusion - induced renal injury not by inhibition of coagulation abnormalities but rather by direct inhibition of leukocyte activation . Another group has also described a combination of anti - inflammatory and anticoagulatory actions of apc during renal ischemia reperfusion that provided microvascular protection . In this model, since leukocytes were, therefore, thought to be of central importance in the development of organ injury, the authors hypothesized that apc inhibits neutrophil adhesion, thereby attenuating the release of neutrophil elastase and oxygen radicals, both of which increase vascular permeability . However, since neither group had the opportunity to evaluate the actions of apc on the microcirculation in vivo, their conclusions remain speculative . The effects of apc on ischemia reperfusion - induced organ damage have also been evaluated in hepatic tissue: yamaguchi and colleagues investigated the role of apc in a model of ischemia reperfusion - induced injury in rat livers . Before inducing liver ischemia by portal vein cross - clamping for 30 min, the serum concentrations of cytokine - induced neutrophil chemoattractants (cinc) were significantly reduced by apc application . Simultaneously, an observed lowering of myeloperoxidase activity in hepatic tissue during apc application indicated a lesser accumulation of neutrophils in the liver . Of interest, an inactive derivative of factor xa, which represents a selective inhibitor of thrombin generation, was also effective in decreasing cinc concentrations and could prevent leukocyte accumulation . This indicated that, in this case, postischemic hepatic microcirculatory disturbances were induced by microthrombotic occlusion in a thrombin - dependent mechanism . Apc has been used in several sepsis models to modulate the sepsis - induced proinflammatory response . Thus, a prospective, blinded, placebo - controlled trial has been performed in a rabbit model of meningococcal endotoxin - induced shock . Strikingly, apc administration could not prevent an endotoxin - mediated decrease in mean arterial pressure and an increase in heart rate . However, despite lacking any effect on macrohemodynamics, survival was significantly improved from 45% to 75% . The authors hypothesized that apc had an effect on the microcirculation since the expected macrohemodynamic changes could not be characterized in their experiment . In a model of endotoxin - induced acute respiratory distress syndrome, apc attenuated endotoxin - induced pulmonary vascular injury, probably by the significant reduction of endotoxin - induced pulmonary leukocyte infiltration . In light of these findings, it is highly likely that apc also has a direct effect on the microcirculation during endotoxemia . However, none of these models allowed the direct visualization of the microcirculation and, thus, the study of apc effects on microcirculatory dysfunction . To evaluate the effects of apc on the microcirculation in vivo during endotoxemia, our group tested apc in a sepsis model that permitted observation of the microcirculation under non - invasive, normotensive conditions . Endotoxin induced a massive increase in leukocyte accumulation in precapillary arterioles and postcapillary venules, with a maximal effect observed after 8 hours (fig . Macrohemodynamic disturbances could be excluded by careful analysis of macrohemodynamic and microhemodynamic parameters in this model . Moreover, the next step in the leukocyte endothelial - cell interaction (namely, leukocyte adherence) was also significantly inhibited in arterioles and venules (fig . In addition, endotoxin - mediated deterioration of microvascular perfusion was counteracted effectively by intravenous apc administration . Since leukocyte rolling is predominantly mediated by adhesion molecules of the selectin family, the reduction of leukocyte rolling by apc may indicate a modulation of selectin expression on leukocytes and the endothelium . Visualization of leukocyte sticking in the arteriole by intravital fluorescence microscopy (performed using a charge - coupled device camera before and after lipopolysaccharide administration). Representative arterioles (a) before and (b) 8 hours after endotoxin application . Note that leukocytes also adhere to the endothelial lining of arterioles during endotoxemia despite high flow conditions . Effect of activated protein c (apc) on lipopolysaccharide (lps)-induced (a) arteriolar and (b) venular leukocyte rolling . The apc group contained animals (n = 8) that received lps (2 mg / kg) and apc (24 g / kg per 8 hours intravenously). The control group contained vehicle - treated animals (n = 7) that received lps (2 mg / kg) only . * p <0.05, apc animals versus lipopolysaccharide - treated controls (unpaired student's t - test). P <0.05, intergroup differences from baseline (paired student's t - test). Effect of activated protein c (apc) on lipopolysaccharide (lps)-induced (a) arteriolar and (b) venular leukocyte adhesion . The apc group contained animals (n = 8) that received lps (2 mg / kg) and apc (24 g / kg per 8 hours intravenously). The control group contained vehicle - treated animals (n = 7) that received lps (2 mg / kg) only . * p <0.05, apc animals versus lipopolysaccharide - treated controls (unpaired student's t - test). P <0.05, intergroup differences from baseline (paired student's t - test). This view corresponds to in vitro data obtained by vascular endothelial cell culturing, where human plasma - derived and human cell - produced protein c blocked e - selectin - mediated cell adhesion to the endothelium . From our in vivo study, we concluded that it is highly likely that a specific anti - inflammatory interaction between apc and the endothelium is responsible for microcirculatory protection . Since thrombin inhibition alone obviously does not reduce endotoxin - mediated leukocyte rolling and adhesion, anticoagulatory apc activities (namely, inhibition of factors va and viiia that would result in less thrombin formation) are probably unlikely to be responsible for the protective effects of apc on the microcirculation during endotoxemia . Table 1 shows an overview of the differential effects of the anticoagulants apc, antithrombin, and hirudin (a specific thrombin inhibitor) on different steps in the leukocyte endothelial - cell interaction and capillary perfusion in septic disorders . Differential regulation of endotoxic microcirculatory disorders by coagulatory inhibitors note that hirudin is not antiadhesive and does not protect against endotoxic capillary dysfunction . The protein c and antithrombin anticoagulant pathways represent major mechanisms in controlling microvascular dysfunction during ischemia reperfusion injury and sepsis . Local and systemic antithrombin and apc apc and antithrombin have been used in experimental models to reduce ischemia reperfusion injury and to improve septic disorders with comparable efficacy . The exact molecular mechanisms of action of apc and antithrombin during ischemia reperfusion and endotoxemia remain unclear . However, it is highly likely that a specific anti - inflammatory interaction with the endothelium and leukocytes is responsible for apc - related microcirculatory protection . Thus, apc treatment using a dose corresponding to that used in the recombinant human activated protein c worldwide evaluation in severe sepsis (prowess) study in vivo reduced the extent of transient leukocyte endothelial - cell interactions (leukocyte rolling) and inhibited subsequent firm leukocyte adherence (leukocyte sticking) to the endothelium . Both mechanisms lessened endotoxin - mediated microvascular perfusion injury . These microcirculatory apc actions may also be used under clinical situations involving microcirculatory disorders . From the majority of experimental studies presented here, there is, however, no evidence to support antithrombotic (anticoagulatory) therapy specifically targeting thrombin inhibition, since thrombin inhibitors (e.g. Hirudin and heparin) do not provide comparable protection of the microcirculation, but, on the contrary, may even aggravate sepsis - mediated disorders . Apc = activated protein c; cinc = cytokine - induced neutrophil chemoattractants; prowess = recombinant human activated protein c worldwide evaluation in severe sepsis.
Temperature is undoubtably one of the most important parameters to know in various fields of science and technology, and in everyday life . Major contactless techniques include optical pyrometry, ir thermography, and phosphor thermometry . Among them, optical pyrometry is only suitable for measurements at high temperatures (> 600 c). On the other hand, for example, precise temperature determination is essential in marine research, geochemical studies,(3) medicine, and aeronautics,(6) to mention only a few areas . Moreover, temperature is a key parameter in optical sensing since the response of virtually all such sensors is temperature dependent . Ir thermography has become very widespread but is still rather expensive and may suffer from various artifacts. (7) luminescent temperature - sensitive materials represent a promising alternative here . Thermographic phosphors are well established and have been in use for decades. (8) some of them find application in commercial devices (e.g., fiber - optic thermometers from lumasense technologies, www.lumasenseinc.com). However, most of the thermographic phosphors are intended for sensing high temperatures and become practically useless at ambient conditions. (9) the suitable phosphors for rt sensing include manganese(iv)-doped magnesium fluorogermanate mg4fgeo6, chromium(iii)-doped yttriumaluminum garnet y3al5o12,(12) aluminum oxide (ruby), spinel mgal2o4,(15) and yttriumaluminum perovskite yalo3,(16) as well as europium(iii)-doped lanthanum oxysulphide la2o2s . Some other phosphors also have been proposed. (19) the limitations of these phosphors include rather low temperature sensitivity and/or insufficient luminescence brightness . Apart from thermographic phosphors, organometallic complexes which possess temperature - dependent luminescence were found to be very useful probes . Recently, they became increasingly popular and were applied in dually sensing materials and pressure - sensitive paints . Such materials usually rely on the use of ruthenium(ii) polypyridyl complexes and europium(iii) tris--diketonates . Generally, these temperature probes suffer from photostability issues, decreased luminescence intensity at higher temperatures, and pronounced cross - sensitivity to oxygen so that their immobilization in gas - blocking polymers such as polyacrylonitrile is usually essential. (32) some temperature - sensitive fluorophores also were reported but they are less convenient due to measurements of fluorescence intensity and not the decay time . Evidently, state of the art thermographic phosphors and luminophores have their limitations and more advanced materials are highly desired . An ideal luminescent material for sensing temperature should fulfill the following requirements: (i) have high brightness; (ii) possess excitation in the visible or nir part of the spectrum to minimize autofluorescence interferences; (iii) have high - temperature sensitivity in the desired range; (iv) be photostable; (v) have negligible cross - sensitivity to other analytes such as, e.g. Oxygen; and (vi) be commercially available or simple to manufacture . Very often relatively long luminescence decay times (> 1 s) are desirable in order to enable lifetime interrogation . In this contribution we will demonstrate that chromium(iii)-doped yttrium aluminum borate (yab) fulfills all these requirements and is, therefore, a thermographic phosphor of choice for sensing and imaging at ambient temperatures . Yttrium oxide (99.99%), gadolinium nitrate hexahydrate (99.9%), chromium(iii) nitrate nonahydrate (99%), and molybdenum(vi) oxide (99.5+%) were obtained from aldrich (www.sigmaaldrich.com), aluminum nitrate nonahydrate (99+%), yttrium nitrate hexahydrate (99.9%), urea (99.5%), and chromium(iii) oxide (99,997%) were from fischer scientific (www.fishersci.com), aluminum oxide (> 99%) was from merck (www.merck.de), hydrogel d4 was from advansourse biomaterials (www.advbiomaterials.com), boron trioxide (> 99%), potassium hydroxide, and potassium sulfate (99+%) were from fluka (www.sigmaaldrich.com), and boric acid (99.99%) was from abcr (www.abcr.de). Microcrystalline powders of chromium(iii)-doped yag y3al4.8cr0.2o12, spinel mgal1.96cr0.04o4, and ruby al1.96cr0.04o3 were obtained from the nitrates and urea with the solution combustion technique . The luminescent beads of ruthenium(ii) tris-1,10-phenanthroline in poly(acrylonitrile) (= ru - phen / pan) and europium(iii) tris(2-thenoyltrifluoroacetonato)-4-(4,6-di(indazol-1-yl)-1,3,5-triazin-2-yl)-n, n - diethylbenzenamine in poly(styrene - block - vinylpyrrolidone) (eu(tta)3deadit / ps - pvp) were prepared as described elsewhere . The cr(iii)-doped yab crystals were obtained by high - temperature flux growth technique the appropriate amounts of the yttrium oxide, aluminum oxide, boron trioxide, and chromium(iii) oxide were homogenized in a mortar and mixed with a flux system consisting of potassium sulfate and molybdenum(vi) oxide . The mixture was loaded in platinum crucibles and melted in a front - loaded electric resistance furnace, equipped with a programmable temperature controller . The temperature program consisted of heating to 1120 c at 300 deg / h, keeping that temperature for 3 h, and then slowly cooling to 900 c at a rate of 1 deg / h . Upon removing the flux by boiling in concentrated potassium hydroxide (c = 8.0 m) yab crystals (100 m10 mm) microcrystalline yab powders were synthesized employing the solution combustion method with urea as a fuel . Typically, 5 mmol (1.915 g) of yttrium nitrate hexahydrate, 13.514.85 mmol (5.0645.570 g) of aluminum nitrate nonahydrate, 0.251.5 mmol (0.1000.600 g) of chromium nitrate nonahydrate, 20 mmol (1.236 g) of boric acid, and 100 mmol (6 g) of urea were dissolved in 10 ml of deionized water . The solution was heated in a resistance furnace to 500 c (heating rate 20 deg / min) and this temperature was maintained for 15 min . The obtained raw material was milled in an agate mortar and sintered in a porcelain crucible for 24 h at 1100 c in air . The powder was ground in a ball mill to result in 2.6 0.5 m microcrystalls . Microcrystalline cr(iii)-doped gadolinium aluminum borate (gab) powder was obtained in a similar manner, where gadolinium(iii) nitrate was used instead of yttrium(iii) nitrate . Emission and excitation spectra were acquired on a hitachi f-7000 fluorescence spectrometer (www.hitachi.com) equipped with a red - sensitive photomultiplier r 928 from hamamazu (www.hamamatsu.com). The emission spectra were corrected for the sensitivity of the pmt as described previously. (38) the emission and excitation spectra were acquired for the phosphor powders fixed in a homemade cell positioned at 60 toward the excitation light . Luminescence phase shifts were measured with a two - phase lock - in amplifier (sr830, standford research inc ., www.thinksrs.com). The phosphors were (crystals or microcrystalline powders) placed in a homemade cuvette and excited with the light of a 405 nm led (www.roithner-laser.com) filtered through a bg-12 glass filter (www.schott.com). The luminescence was detected with a photomultiplier tube (h5701 - 02, hamamatsu, www.sales.hamamatsu.com) after passing an rg 630 filter from schott . A bifurcated fiber bundle was used to guide the excitation light to the probe and to guide back the luminescence . Modulation frequencies of 1 and 2 khz were used for cr(iii)-doped yab and cr(iii)-doped gab, respectively . The luminescence decay profiles were acquired on a bmg labtech fluostar optima microplate reader (www.bmg-labtechnologies.com). The powder was excited at 405 nm and a long - pass rg 9 filter was used for the emission . Imaging in the time domain was performed with a gated sensicam camera (cooled ccd - chip, 640 480 pix, black and white, www.pco.de). A high - power 405-nm led (www.roithner-laser.com) was used as an excitation source . The x - ray powder diffraction data were collected with a laboratory powder diffractometer (philips x - pert) from a flat sample (braggbrentano geometry) with use of cu k radiation . The measurements were carried out from 10 to 95 2, with a step width of 0.02 and a constant counting time of 5 s per step . Was taken from icsd, fiz karlsruhe (yal3(bo3)4, 91962,(41) ybo3, 84653). (42) a pseudo - voigt profile shape function was used . Yttrium aluminum borate (yab) is isostructural with the mineral huntite and has a trigonal crystal structure of r32 space group. (43) both trivalent rare - earth and transition metal ions (e.g., chromium(iii)) can be doped into the material, where rare - earth ions then occupy yttrium sites and transition metal ions replace aluminum(iii) on sites of octahedral symmetry. (44) it is known that concentration quenching of luminescence in this structure is usually lower than in other solid hosts, thus allowing higher dopant levels. (45) two different techniques were used to obtain cr(iii)-doped yab, namely flux growth and the solution combustion method . In the first method, cr(iii)-doped yab crystals are grown from the solution in high - temperature flux of potassium molybdate . The yab crystals obtained by this method have high purity as demonstrated by powder diffraction patterns (figure 1). First, the method is time - consuming due to slow cooling required during crystal growth (1 deg / h) and has a laborious extraction procedure (dissolving of the flux in concentrated potassium hydroxide). Second, micrometer - sized powders are strongly preferred for practical applications and grinding of the crystals becomes necessary . Third, the amount of dopant cannot be controlled precisely since some of the chromium(iii) ions may remain in the flux . Therefore, the solution combustion method represents a promising alternative to the flux growth technique . Microcrystalline powders are easily obtained by combustion of the solutions containing respective metal nitrates, boric acid, and urea (used as a fuel) with subsequent 24 h sintering at 1100 c . The powder diffraction patterns (figure 1) the impurity is attributed to the presence of about 4% yttrium borate(48) quantified by rietveld refinement . In fact, maia et al. (49) have shown that yabs obtained by the solgel and the polymeric precursor methods contain ybo3 phase as an impurity . The authors have demonstrated that it is possible to obtain pure microcrystalline powders using the polymeric precursor method with subsequent sintering at 1150 c . Powder diffraction patterns for yabs: (a) obtained by the combustion method with subsequent 24 h sintering at 1100 c, impurity of about 4% ybo3; (b) flux growth crystals (5% cr(iii) in the oxide mixture), powder pattern shows preferred orientation; (c) simulated powder diffraction pattern of single phase yab . The emission and luminescence excitation spectra for the cr(iii)-doped yab are shown in figures 2 and 3, respectively . The phosphor has an intense broad luminescence in the nir region associated with the t2 a2 transition and an additional sharp r - line associated with the e a2 transition . The schematic configuration coordinate diagram (see figure 4b) highlights the zero - phonon nature of the sharp transition from the e state as compared with the broad vibronic transition from the t2 state, which is placed toward lower energy . In the tanabesugano diagram for cr(iii) in octahedral crystal fields (see figure 4a) the 10 dq / b values for all investigated cr(iii) containing phosphors are indicated . All hosts provide 10 dq / b ratios which have the e state as the lowest lying emitting state . It is evident that for the yab the energy difference between the two emitting states (e and t2) is the smallest . Excitation of the cr(iii)-doped yab is attributed to the a2 t2 and a2 t1 transitions with band maxima at 421 and 599 nm, respectively . Therefore, the cr(iii)-doped yab can be excited both in the blue and in the red part of the spectrum, which is nicely compatible with the emission of the 635 nm red laser diode . Notably, among thermographic phosphors only cr(iii)-doped y3al5o12 shows similar spectral features (figure 3). It is evident that other thermographic phosphors such as cr(iii)-doped mgal2o4 and al2o3 as well as mn(iv)-doped mg4fgeo6 cannot be efficiently excited in the red part of the spectrum (figure 3). The 10 dq / b values of yal3(bo3)4:cr (solid), y3al5o12:cr(dotted), mgal2o4:cr (dot and dash), and al2o3:cr (dashed) are indicated . (b) configuration coordinate diagram (not to scale) of cr(iii) in octahedral environments . Figure 5 shows the dependences of the luminescence decay time and the luminescence intensity on the amount of cr(iii) ions which substitute al(iii) in yab . Evidently, concentration quenching is observed even at rather low cr(iii) doping levels, which is indicated by the decrease in luminescence decay time . However, the luminescence intensity does increase with increasing of cr(iii) doping since more light can be absorbed by the phosphor . At higher levels of the activator concentration quenching becomes predominant and the intensity decreases after more than 4% of aluminum ions are substituted by chromium (x = 0.12). Thus, yabs where 24% of al(iii) is substituted by cr(iii) can be a good compromise between luminescence brightness and the degree of concentration quenching . The luminescence decay time of cr(iii)-doped yab was found to be highly temperature - dependent, which is the basis for its use as a thermographic phosphor (figure 6). Generally, the crystals obtained by top - seeded solution growth show the longest decay times and the highest temperature sensitivity approaching 1% of the lifetime change per 1 deg . For all cr(iii)-doped yabs, temperature sensitivity is the highest at 0 c and becomes lower if the temperature increases . The decay time becomes slightly shorter if the crystals are ground into powder (figure 6). This common phenomenon originates from inner defects in the crystal structure which accumulate during grinding . Notably, the lifetime for the powders obtained via the combustion route is generally lower than that for the crystals and is less temperature - dependent, which is attributed to the impurity of yttrium borate and inner defects in the crystal structure . As demonstrated by maia et al.,(49) yabs of higher purity can be obtained using the polymer precursor method . Such yabs are likely to show improved photophysical performance (longer decay times, higher brightness, and temperature sensitivity). Temperature dependence of the luminescence decay time for cr(iii)-doped yab phosphors: (1) crystals obtained by high - temperature solution growth (5% cr(iii) in the oxide mixture); (2) ground crystals; and (3, 4, 5) microcrystalline powders obtained via combustion with 1%, 2.5%, and 4% of cr(iii), respectively . Evidently, temperature significantly affects the intensity of the r - line associated with the e a2 transition, which decreases with temperature . On the other hand, broad nir luminescence associated with the t2 the isosbestic point at 738 nm is explained by the thermal population of the t2 emitting state from the metastable e state . As has been shown in figure 4 for the yab host these two states are closest in energy . As demonstrated by dominiak - dzik et al.,(50) at lower temperatures (77 k) the broad nir emission further decreases in intensity accompanied by the increase of intensity for the r - line . Notably, the overall decrease in luminescence intensity on going from 1 to 70 c is negligible and does not exceed 0.2% . Thus, the s / n ratio for cr(iii)-doped yab remains constant in this temperature range . On the contrary, if luminescent metalligand complexes are used as temperature probes the decrease in luminescence intensity is usually very pronounced and is often as high as several percent per degree. (34) this results in much lower s / n ratios at higher temperatures . Despite the fact that cr(iii)-doped yab emits from both e and t2 states, the luminescence decay profiles are virtually monoexponential (correlation coefficient> 0.9996), figure 8 . The decay times obtained in the time domain and in the frequency domain are virtually identical, e.g. 154 and 155 s for the yab containing 2.5% cr(iii), for the time domain and frequency domain measurements, respectively . Temperature dependence of the luminescence intensity for cr(iii)-doped yab (2.5% cr(iii), combustion method). Time dependence of the emissions for cr(iii)-doped yab (2.5% cr(iii), combustion method). Lines represent a fit via monoexponential decay model (main graph) and a linear fit (the insert). The luminescence brightness of the cr(iii)-doped yab microcrystalline powder (2.5% cr(iii)) was estimated from the corrected emission spectrum under 405 nm excitation . It was compared to the brightness of other thermographic phosphors which are also excitable at this wavelength (figure 3). For all the phosphors, 20 mg of a microcrystalline powder (25 m) was dispersed in 1 ml of poly(ethylene glycole) and the suspension was interrogated in a fluorescence spectrometer . The relative brightnesses were determined to be 1, 0.61, 0.69, 0.42, and 0.16 for mn(iv)-doped mg4fgeo6, cr(iii)-doped yal3(bo3)4, al2o3, mgal2o4, and y3al5o12, respectively . In another experiment, the microcrystalline phosphor powders were positioned in a homemade cell and excited in the fluorescence spectrometer in a 60 angle, at two different temperatures, 1 and 60 c . These results are summarized in table 1 . As can be seen, they are similar to those obtained for the phosphor suspensions . Evidently, cr(iii)-doped yab shows excellent brightness, which is comparable to that of mn(iv)-doped mg4fgeo6 (lamp phosphor). Unfortunately, comparison with the established temperature - sensitive organometallic complexes is more complicated due to scattering of microcrystalline powders . Therefore, freeze - dried polymeric beads of ru - phen in polyacrylonitrile and eu(tta)3deadit in poly(styrene - block - vinylpyrrolidone) were used for comparison . At low temperature (1 c) the brightness of these materials is comparable to that of the cr(iii)-doped yal3(bo3)4 . However, in contrast to thermographic phosphors, the brightness of the organometallic complexes deteriorates dramatically if the temperature is increased to 60 c (table 1). For ru - phen as is clearly visible from figure 9, cr(iii)-doped yabs shows much higher temperature sensitivity compared to other visible light - excitable thermographic phosphors such as cr(iii)-doped mgal2o4,(15) cr(iii)-doped al2o3,(14) and mn(iv)-doped mg4fgeo6. (11) it should be emphasized that although other thermographic phosphors such as ruby- and mn(iv)-doped mg4fgeo6 are rather established in thermometry, they provide 35 times poorer resolution that cr(iii)-doped yab . At higher temperatures cr(iii)-doped y3al5o12 could compete with yabs in terms of temperature sensitivity, but its performance is compromised by low brightness . As can be seen, ruthenium(ii) tris-1,10-phenanthroline complex (ru - phen) has lower temperature sensitivity than cr(iii)-doped yab . Generally, only luminescent europium(iii) complexes such as a visible - light excitable eu(tta)3deadit(33) possess higher temperature sensitivity exceeding 1% per deg . Moreover, the organic metalligand complexes need to be embedded into gas blocking polymers such as polyacrylonitrile, poly(vinylidene chloride - co - acrylonitrile), etc . In order to avoid quenching by oxygen . This is not necessary for the phosphors which additionally benefit from excellent photostability and can be continuously excited with leds for many days and even weeks without showing any deterioration . On the contrary, all metal complexes are prone to photobleaching, which is often very pronounced for the europium(iii) luminophores . Table 1 summarizes the properties of the most common probes used in room temperature thermometry . It is evident that the cr(iii)-doped yab is an excellent alternative to the state of the art thermographic phosphors and luminescent organometallic complexes . Temperature sensitivities of the visible light - excitable thermographic phosphors and luminescent metalorganic complexes: (1) eu(tta)3deadit in polystyrene; (2)yal3(bo3)4:cr crystals obtained by high - temperature solution growth (5% cr(iii) in the oxide mixture); (3) yal3(bo3)4:cr microcrystalline powder (2.5% cr); (4) y3al5o12:cr; (5) ru - phen in polyacrylonitrile; (6) mgal2o4:cr; (7) al2o3:cr; and (8) mg4fgeo6:mn . Pressure - sensitive paints, particularly, rely on luminescent indicators which are used to monitor the total pressure via oxygen partial pressure. (31) unfortunately, temperature affects both the luminescence of an oxygen indicator and gas permeability coefficients of a polymer where the indicator is dissolved . To obtain correct it is usually achieved by addition of temperature - sensitive metal complexes (commonly immobilized in beads) or thermographic phosphor powders to the pressure - sensitive paint . Figure 10a shows a photographic image of the sensor coating (cr(iii)-doped yab dispersed in hydrogel d4), which reveals a very homogeneous layer . This is of course not surprising considering the size of the microcrystalline powder (2.6 0.5 nm). The luminescence decay times (figure 10bd) calculated from the images obtained with a gated ccd camera clearly demonstrate the suitability of the material for imaging of temperature distribution on surface . However, it should be considered that the sensitivity of the ccd cameras decreases in the nir region, which decreases the s / n ratio in the case of cr(iii)-doped yab . (a) a photographic image of the 10 m thick coating (50% w / w of cr(iii)-doped yab, 2.5% doping, in hydrogel d4). (bd) lifetime distribution for the same coating at different temperatures . Apart from yttrium aluminum borate several similar hosts were reported . It is well - known that such trivalent ions as lanthanum, gadolinium, and lutetium can occupy the yttrium sites and scandium(iii) or gallium(iii) can substitute aluminum . Thus, fine - tuning of photophysical properties of the cr(iii)-doped borate phosphors might be possible . For example, it was demonstrated(44) that cr(iii)-doped yttrium scandium borate is excitable at longer wavelength than cr(iii)-doped yab and has bathochromically shifted emission . Both excitation and emission spectra are bathochromically shifted if aluminum is partly substituted by gallium. (47) for both phosphors the intensity of the sharp r - line associated with the e a2 transition is very low . Our preliminary investigations indicate that cr(iii)-doped ysc3(bo3)4 and yga3(bo3)4 possess lower brightness than the respective cr(iii)-doped yal3(bo3)4 with the same doping level . On the other hand, the brightness of cr(iii)-doped gadolinium aluminum borate (gab) is comparable to that of cr(iii)-doped yab . The excitation spectra of the two phosphors are very similar (exc 423 and 591 nm for gab and 421 and 600 nm for yab). However, the luminescence decay time of cr(iii)-doped gab is significantly lower than that of cr(iii)-doped yab (figure 11). Thus, this phosphor may be suitable for application in dual lifetime referenced (dlr) optical sensors(51) since inertness to chemical and physical parameters (including temperature) is highly desirable in this method . Temperature dependence of the luminescence decay time for cr(iii)-doped yal3(bo3)4 and gdal3(bo3)4 microcrystalline powders (2.5% cr(iii) doping). In conclusion, cr(iii)-doped yab represents a new type of thermographic phosphor which provides uncompromised solution for sensing and imaging of ambient temperatures . The new material is distinguishable for the following features: (i) excitation in the blue and the red part of the spectrum and compatibility with red laser diodes; (ii) nir emission; (iii) excellent luminescence brightness; (iv) large temperature coefficients of the decay time at ambient temperatures; (v) uniform brightness in the whole temperature range of interest; and (vi) simplicity in preparation via the combustion route . Compared to luminescent metalligand complexes, cr(iii)-doped yab additionally benefits from chemical and photochemical inertness and the absence of the cross - sensitivity to oxygen . Potential applications include sensing and imaging of temperature in those fields where invasiveness is undesirable and combination of the phosphor with other indicators in temperature - compensated optical chemosensors and pressure - sensitive paints.
About 85% of lung cancers are non - small - cell lung cancers (nsclcs), with lung adenocarcinoma (ladc) and lung squamous cell carcinoma (lscc) as the major subtypes of nsclc.1, 2 although encouraging targeted therapies have dramatically afforded benefits to patients with ladc, few therapeutic targets have been validated for patients with lscc.3, 4, 5 to date, the treatment options for patients with lscc are limited, and platinum - based chemotherapy is still the gold standard for first - line treatment; however, some lsccs are intrinsically resistant to chemotherapy . Virtually all patients, including initial responders, rapidly develop chemoresistance,6, 7, 8, 9 which, in the absence of alternative therapies, leads to an overall 5-year survival rate of less than 20% . Therefore, a better understanding of the molecular determinants of lscc resistance to chemotherapy is a critically important step to overcome the clinical chemoresistance currently responsible for the dismal survival rates of patients with lscc . We recently reported, for the first time, that piwi - interacting rna likes (pir - ls), a type of functional small non - coding rnas (sncrnas), played critical roles in nsclc tumorigenesis and differentiation . We have profiled pir - ls expression in normal human bronchial epithelial (hbe) and nsclc cells, and we found that lscc, ladc, and hbe cells could be clearly clustered together on the expression patters of all pir - ls . We also found that pir - ls were aberrantly differentially expressed in nsclc and hbe cells as well as between lscc and ladc subtypes . Using filtered log fold change = 1 and false discovery rate <0.05 as criteria, we identified that there were seven pir - ls that could distinguish lscc from both ladc and hbe cells, indicating that these seven pir - ls were lscc - related pir - ls . In this study, we observed the effects of lscc - related pir - ls in resistance caused by cisplatin (cddp)-based chemotherapeutic agents commonly used for patients with lscc . Characteristically, we identified that pir - l-138 was upregulated upon cddp - based chemotherapy both in vitro and in vivo, and, further, we found that targeting the upregulated pir - l-138 resulted in enhanced sensitivity of lscc to cddp - based chemotherapy both in vitro and in vivo, indicating that pir - l-138 is a key contributor to cddp - based chemoresistance and that targeting pir - l-138 could be a potential strategy to overcome chemoresistance for patients with lscc . To determine whether these lscc - related pir - ls played roles in chemoresistance, we analyzed the relationships between their expression levels and the sensitivities to gold standard first - line platinum - based chemotherapy . Given that cddp is the backbone of platinum - based chemotherapy, which is defined as a combination of a platinum compound (cisplatin or carboplatin) and one of the four chemotherapy agents (gemcitabine, docetaxel, paclitaxel, and vinorelbine), we evaluated expression levels of lscc - related pir - ls to these agents commonly used for patients with nsclc . We observed that lscc - related pir - ls were upregulated upon exposure to agents (figure s1). To further identify whether these pir - ls were lscc - related sncrnas, we also observed both lscc - related and non - lscc - related pir - ls in four lscc as well as in four ladc cell lines (figure s1), and pir - ls levels were measured in cells treated with cddp, gemcitabine, docetaxel, and their corresponding control solutions (pbs or dmso) at a drug concentration required for 50% inhibition (ic50) for 24 hr (table s1; figure s2). Among pir - ls, upon exposure to these chemotherapeutic agents, we observed a significant consistent and substantial increase of pir - l-138 levels in all four lscc cells treated with cddp, gemcitabine, and docetaxel (figure 1a), indicating that pir - l-138 was upregulated upon cddp - based chemotherapy in vitro . In contrast, neither pir - l-138 in all four ladcs nor any non - lscc - related pir - ls in all four lsccs were observed to show an obviously consistent and substantial increase upon cddp, gemcitabine, or docetaxel treatment (figure s1), indicating that the upregulated pir - l-138 was lscc - related, which was consistent with the data obtained using rna sequencing (rna - seq) (figure 1b) as we reported previously . Together, these results suggest that lscc - related pir - l-138 is upregulated upon cddp - based chemotherapy in vitro . To determine whether the upregulated pir - l-138 upon cddp - based chemotherapy was a cell culture artifact, we analyzed its expression levels in patient - derived xenograft (pdx) tumors developed from tissues of patients with lscc and ladc, which were used in our previously published study . The pdx tumors were from untreated mice or mice treated with cddp - based chemotherapy (cddp plus gemcitabine) for 8 and 28 days, respectively . We observed that pir - l-138 levels were significantly increased in the tumors derived from lscc tissues (patients umb410 and mda2131 - 11) at both time points after the mice had been treated with cddp - based chemotherapy for 8 and 28 days (figures 1c and 1d). On the contrary, pir - l-138 showed an obvious downregulation in tumors derived from ladc tissues (patient mda2131 - 1) at both time points after the mice had been treated with cddp - based chemotherapy for 8 and 28 days (figure 1e), suggesting that pir - l-138 is induced upon cddp - based chemotherapy in tumors developed from lscc in vivo . Next, we wanted to identify the roles of the upregulated pir - l-138 in chemoresistance of lscc . Given that cddp is the backbone agent of platinum - based chemotherapy for patients with nsclc, we used cddp as the representative agent to verify roles of pir - l-138 in chemoresistance . To determine whether the cddp - induced pir - l-138 played functional roles in vitro, we used dna oligonucleotides that were complementary to pir - l-138 as antisense (anti-138) for blocking pir - l-138 because the crna oligonucleotides could trigger a short interfering rna (sirna)-like response.10, 12 targeting pir - l-138 followed by cddp treatment in two lscc representative cell lines of h157 and skmes-1, we found that blocking pir - l-138 resulted in decreased cell viability (figure 2a), which was caused by an increased apoptotic cell population represented by a substantial increase of the sub - g1 fragment (figure 2b; figures s3a h157 and skmes-1 cells treated with anti-138 showed a significantly increased percentage of annexin v - positive cells, cleaved capase-3, and cleaved poly adp ribose polymerase (parp) (figure 2c; figures s4a s4c), further confirming that targeting pir - l-138 increases the sensitivity of lscc cells to cddp in vitro . To further confirm the roles of cddp - induced pir - l-138 in apoptosis, we used a tumor xenograft model to determine whether targeting pir - l-138 could enhance cddp - induced cell apoptosis of tumors developed from h157 cells inoculated in nu / nu mice . H157 cells were inoculated into mice, tumors at least 5 mm in diameter were considered established, and all tumors were confirmed by pathology examination (figure 3a, right). An intraperitoneal (i.p .) Cddp injection was given on day 1, followed by an intratumoral (i.t .) Injection of scr or anti-138 oligonucleotides formulated with maxsuppressor in vivo rnalancerii on days 3, 7, 10, 14, and 16, respectively . Compared with tumors formed in untreated mice and in mice treated with cddp plus scr, the tumors in mice treated with cddp plus anti-138 were significantly smaller (figure 3a, left and center and growth curves), indicating that targeting pir - l-138 inhibited tumor growth . Further, compared with tumors treated with cddp and scr control, tumors treated with cddp plus anti-138 exhibited higher expression of cleaved caspase 3 (figure 3b), indicating that targeting pir - l-138 enhanced cddp - induced cell apoptosis in tumors . In addition, we found that tumor areas that showed high concentrated anti-138 also showed high levels of cleaved caspase-3 and cleaved parp (figures 3c and 3d), further conforming that blocking cddp - upregulated pir - l-138 accelerates cddp - induced apoptosis of tumors derived from lscc cells . Finally, we wanted to decipher the potential mechanisms underlying cddp - induced pir - l-138 in chemoresistance . Given that cddp - induced pir - l-138 inhibits apoptosis and that these two lscc - representative cell lines harbor p53 mutations, we hypothesized that pir - l-138 could be involved in regulating functions of mouse double minute 2 homolog (mdm2) because mdm2 and its three isoforms (90, 75, and 60 kda) have been implicated in p53-independent apoptosis as well as in chemoresistance.15, 16, 17 to test our hypothesis, we measured the expression levels of total mdm2 and its cleaved isoforms after blocking and ectopic expression of pir - l-138, respectively . As shown in figure 4a, blocking pir - l-138 increased full - length mdm2 and decreased the 60-kda cleaved isoform (referred to hereafter as p60-mdm2) upon cddp treatment, and, in contrast, ectopic expression of pir - l-138 decreased total mdm2 and increased p60-mdm2 upon cddp treatment (figure 4b), indicating that the role of pir - l-138 in apoptosis inhibition was related to p60 mdm2 . Strikingly, however, we observed that reduced p60-mdm2 was accompanied with an increase in its serine-166 phosphorylation and that increased p60-mdm2 was accompanied by a decrease in its serine-166 phosphorylation (figures 4a and 4b). Based on the positive relationship between pir - l-138 and p60-mdm2, a negative correlation with serine-166-p60-mdm2, and our experiences with this type of sncrnas, we hypothesized that pir - l-138 could directly bind to p60-mdm2 to play roles in apoptosis . To determine whether pir - l-138 directly interact with p60-mdm2, biotin - conjugated pir - l-138 rna oligonucleotides were used as bait to pull down its potential binding proteins in cddp - treated h157 and skems-1 cells . We found that p60-mdm2 was readily detectable in the pull - down products using bio - pir - l-138 but not in the products of bio - scr rna control (figure 4c), indicating that pir - l-138 bound to p60-mdm2 . To confirm the interaction of pir - l-138 and p60-mdm2, we performed an immunoprecipitation (ip) assay using an anti - p60-mdm2 antibody followed by rt - pcr using primers specific for pir - l-138 . Although pir - l-138 was not detectable in the ip products of a control igg antibody, pir - l-138 was easily detected in the products using the anti - p60-mdm2 antibody (figure 4d). To further validate the interaction between pir - l-138 and p60-mdm2 at the individual cell level, we performed a fluorescence in situ hybridization (fish) assay using a digoxin (dig)-labeled dna probe complementary to pir - l-138 and anti - p60-mdm2 antibody . We first tested the probes in hbe4 cells, which express a high level of pir - l-138, to ensure the sensitivity and specificity of the pir - l-138 probe and p60-mdm2 antibody (figures 1b and 4e), and we then measured their distribution upon cddp treatment . We found that both pir - l-138 and p60-mdm2 were predominantly co - localized at the perinuclear area when unchallenged and showed an enhanced and polarized perinuclear pattern upon cddp treatment in both h157 and skmes-1 cells (figure 4e). Together, these results suggest that pir - l-138 directly interacts with p60-mdm2 in response to cddp treatment in p53-mutated lscc h157 and skmes-1 cells . Piwi - interacting rnas (pirnas) have been identified to play roles in transposon silencing, heterochromatin modification, and germ cell maintenance.18, 19 because of our limited knowledge regarding their biogenesis, most studies focused on their functions in the renewal of germline cells and development.20, 21, 22, 23 their potential roles in somatic tissues, particularly in pathogenesis such as tumorigenesis, although poorly studied, have recently been recognized.10, 18, 19, 24 in our recently published study, we demonstrated the presence of a type of functional sncrna class that has certain similarity with pirnas but is expressed in somatic lung bronchial epithelial cells, and we termed them pir - ls . Importantly, pir - ls not only showed differentially expressed patterns among normal hbe, lscc, and ladc cells but also played key roles in fundamental biological activities of hbe, lscc and ladc cells by directly binding to their targeted phosphorylated proteins, suggesting a biological role of pirna - ls in lung tumorigenesis and, possibly, as deterring factors of cellular behaviors for individual tumors . Because little has been studied regarding this type of sncrnas, in this study, we explored the potential effect of pir - ls in chemoresistance to the gold standard first - line platinum - based chemotherapy for treating patient with nsclc . Based on the expression levels of a pir - l that could distinguish a certain type of lung cancer from all other subtypes, we optimized a panel of lscc - related and ladc - related pir - ls, respectively . Our initial intention was to identify an association between these lscc-/ladc - related pir - ls and sensitivity to the chemotherapeutic agents commonly used for patients with nsclc . We first focused on lscc because few therapeutic targets are validated for patients with lscc . We observed some relationships between pir - ls and the sensitivities to agents; characteristically, pir - l-138 was found to be consistently and substantially upregulated after cddp - based chemotherapy treatment in lscc cell lines . The results that pir - l-138 was significantly downregulated in lscc, could distinguish lscc from both hbe and ladc cells, and was consistently upregulated by cddp - based agents suggested that pir - l-138 might play a critical role in cddp - based chemotherapy in lscc . It is important to note that the upregulation of pir - l-138 was not only observed in lscc cell lines but also in the pdx lscc models treated with a cddp - based regimen, indicating that the effect also occurs in lscc tissues in vivo . Our results clearly showed that the upregulated pir - l-138 was critical in protecting lscc cells from cddp - induced apoptosis and that inhibiting pir - l-138 enhanced cddp - mediated apoptosis, suggesting targeting pir - l-138 as a strategy to boost cddp efficacy for patients with lscc . However, a major challenge of using a nucleotide - based treatment strategy is the delivery . Our study provides a proof - of - concept result for the potential use of anti-138 to enhance cddp - mediated apoptosis of lscc cells in vivo because we were using local injection with a sub - optimal dose, which produced only modest anti - tumor activity . We were, therefore, focusing on determining whether anti-138 increased apoptosis at the sites of anti-138 injection by analyzing tumor tissue sections for cleaved caspase-3 and cleaved parp, which are indicators of apoptosis . The concept was supported by the close relationship between higher levels of cleaved caspase-3 and cleaved parp and higher concentrated anti-138 (figure 3). We identified a mechanism underlying chemoresistance caused by cddp - based chemotherapy for patients with lscc, where the functional pir - l-138 is upregulated by cddp - based agents and interacts with p60-mdm2 to inhibit apoptosis, and targeting its upregulation increases lscc sensitivity to chemotherapy agents, indicating that pir - l-138 is a key contributor to cddp - based chemoresistance and that targeting pir - l-138 could be a strategy to overcome chemoresistance for patients with lscc . In our previous study, we have shown that pir - l-163 could directly bind and regulate phosphorylated protein activities . In this study, we explored the possibility that pir - l-138 directly interacts with the p60-mdm2 fragment upon cddp treatment in lscc cells . The direct interaction between pir - l-138 and p60-mdm2 was demonstrated by immunoprecipitation, pir - l-138-specific pull - down, and immunofluorescence co - localization assays . Although p60-mdm2 was reported to be induced in p53-wild - type,25, 26, 27, 28 we identified that the upregulated pir - l-138, upon cddp - based chemotherapy, inhibited cddp - induced apoptosis by interacting with the p60-mdm2 in p53-mutated lscc cells . Strikingly and interestingly, we found that there was a negative correlation between p60-mdm2 and its serine-166 phosphorylation (figure 4a), indicating the possibility that the interaction of pir - l-138 and p60-mdm2 could inhibit its phosphorylation to enhance apoptosis . Also, there could be a totally unknown mechanism underlying p60-mdm2 and serine-166-p60-mdm2 in regulating apoptosis by chemotherapy in p53-mutated lscc cells . In addition, further studies are necessary to determine how pir - l-138 affects p60-mdm2 phosphorylation . For detailed information about all cell lines used here, please refer to our previous publication . All cell lines were recently genotyped for their authentication and routinely treated to prevent mycoplasma growth . Cell transfection was performed as described previously.10, 29, 30 in brief, transfection experiments were performed by using opti - mem medium and lipofectamine 3000 (invitrogen) for dna oligonucleotides or lipofectamine rnaimax (invitrogen) for rna oligonucleotides according to the manufacturer s instructions . Sncrna extraction, rt - pcr, and real - time pcr were performed as described previously . In brief, we used adaptors for sncrna ligation, genome - specific primer (gsp) for reverse transcription, and specific primers for target pir - ls amplification . An adaptor (/5rapp/5-ctgtaggcaccatcaat-3/3ddc/) with both 5 and 3 modification, gsp (5-caagcagaagacggcatacgaattgatggtgcctacag-3), a common reverse primer (5- caagcagaagacggcatacga-3), and a pir - l-138-specific forward primer (5- actttagctctagaattactctgagac-3) were used . The amplification conditions were as follows: denaturation at 95c for 30 s (5 min for the first cycle), annealing at 60c for 20 s, and extension at 72c for 20 s in 25 cycles for rt - pcr and in 40 cycles for real - time pcr . A stock solution of cisplatin (sigma) was dissolved in pbs to a concentration of 1.7 mmol / l (0.5 mg / ml), a stock solution of docetaxel (enzo life sciences) was dissolved in dmso to a concentration of 10 mol / l (8 g / ml), and a stock solution of gemcitabine (gimzar, lilly) was dissolved in pbs to a concentration of 10 mmol / l (3 mg / ml). Cell viability and ic50 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt, life technologies) assay as performed previously,10, 29, 30 and ic50 was defined as the drug concentration that was required for 50% inhibition . To avoid triggering uncertain sirna effects,10, 12 we used complementary dna oligonucleotides as antagonists targeting pir - l-138 . The sequence was 5-aggtctcagagtaattctagagctaaagt-3, and the corresponding scrambled sequence was 5-gataccagggacatacgcttgatcctagc-3. Transfection experiments were performed by using opti - mem medium and lipofectamine 3000 (invitrogen), and the final concentration was 100 nm . Cell cycle and apoptosis analyses were performed as described previously.10, 29, 30 in brief, cell cycle distribution and fluorescein isothiocyanate (fitc) annexin v / pi (bd pharmingen) for the apoptosis assay were analyzed by flow cytometry (becton dickinson), and dna content and percentage of apoptosis cells were analyzed on a facscan flow cytometer in combination with bd lysis software (becton dickinson). Primary antibodies used were as follows: anti - cleaved parp (1:1,000, clone asp214, catalog no . 9541s, cell signaling technology), anti - mdm2 (1:200, clone n-20, lot l099, catalog no . 813, cell signaling technology), anti - p21 (1:200, clone h-164, catalog no . 756, cell signaling technology), anti - phospho - foxo3a (1:1,000, clone ser253, catalog no . A228, sigma - aldrich), human / mouse cleaved caspase-3 (asp175) antibody (1:1,000, catalog no . 31460, pierce).10, 11, 29, 30 protein extraction from cells was performed as we did before, and proteins were extracted from formalin - fixed, paraffin - embedded (ffpe) tissues using the qproteome ffpe tissue kit (catalog no . 37623, qiagen) according to the instructions . We followed the steps for sncrnas pull - down analysis and ip as we did previously, and the specific sequences were as follows: pir - l-138 /5biosg / aggtctcagagtaattctagagctaaagt-3control /5biosg / gauaccaaggacauacgcuuaugcaugcua-3. Immunohistochemistry (ihc) was performed as we described previously.10, 11 tumor samples were fixed in 10% neutral formalin and embedded in paraffin, and sections were prepared less than 5 mm thick, deparaffinized in xylene, and rehydrated . For h&e staining, slides were processed according to our standard protocols . Cell chamber slides (fisher scientific) were fixed with 4% paraformaldehyde (pfa) for half an hour at room temperature (rt), and fissure slides were deparaffinized and rehydrated using standard protocols.10, 11 slides were incubated with 40 g / ml protein kinase k (roche) at 37c for 5 min, followed by a termination reaction in 0.2% glycine (affymetrix usb) for 1 min before processed to pre - hybridization buffer (50% formamide, 1 saline - sodium citrate (scc), 0.5 g / ml yeast rna, and 1 denhardt solution) at 40c for 2 hr . Probes for pir - l-138 and anti-138 were as follows: pir - l-138: /5dign / actttagctctagaattactctgag-3. We added hybridization buffer (enzo) with dig - labeled probes and incubated this overnight at 37c . (1:500) or cleaved caspase3 (1:1,000) at 4c overnight after washing the slides in 2 scc, 1 scc, and 0.2 scc for 10 min, followed by blocking in phosphate - buffered saline tween 20 (pbst) with 5% goat serum (vector laboratories) at rt for 2 hr . Then we incubated the slides in 2.5% goat serum with dylight 594 anti - dig (1:500, catalog no . Di-7594, vector labs) or fitc anti - rabbit (1:750, catalog no . We sealed the slides using mounting medium with dapi for fluorescence (vector labs) and prepared them for scanning . For images, we used zeiss lsm 700 for scanning the slides with the hbe4, h157 and skmes-1 cell lines, tumor tissues slides were scanned by a cytation 3 cell imaging multi - mode reader (biotek instruments) and analyzed biotek s new gen5 software . The high and low staining was distinguished by pixels of 20,000 as the cutoff, and 20 paired lesions were calculated . Tumor tissues used to determine pir - l-138 expression levels were from patient - derived nsclc xenograft models we already prepared in our previous study . For xenograft mouse models, 5 10 h157 cells in 100 l culture medium were inoculated into the lower back and anterior chest of 6- to 8-week - old female athymic nude (nu / nu) mice, which are defective for development of thymic epithelium and lack cell - mediated immunity, according to the protocols approved by institutional animal care and use committees (iacuc no.120901). The tumor sizes were measured twice a week, and tumor volumes were calculated using the formula (/6) length (l) width (w) height (h) of tumors, where l and w represent the longest and shortest tumor dimensions, respectively . Each established tumor model was confirmed by pathology examination . Cddp (1 mg / kg body weight / week) injection was given on day 1, followed by an i.t . Injection of a scramble or anti-138 dna oligo formulated with maxsuppressor in vivo rnalancerii (bioo scientific) at 3 g/20 l on days 4, 7, 11, and 14, respectively . Six mice were used for each group, and the treatments lasted 16 days . Statistical analysis was performed with graphpad prism 6 (graphpad). Significance of the difference between groups was calculated by student s unpaired t test as we did before10, 11, 29, 30 and by two - way anova (tukey s and bonferroni s multiple comparisons test). The results are reported as mean sem, and p <0.05 is considered statistically significant . All authors reviewed, participated in revision, and approved the final version of the manuscript.
Quinine is an alkaloid toxin found in the bark of the south american cinchona (quina - quina) tree . Quinine and its derivative compounds have been used since the 1800s as a homeopathic remedy for analgesia and also as an effective treatment for malaria . Despite its proven efficacy as a schizonticidal agent against the malaria parasite plasmodium falciparum during its intraerythrocytic phase, quinine suffers from a number of contraindications which have made it problematic as an effective therapeutic including a low therapeutic index and high incidence of adverse side effects . Despite this, and a lack of efficacious alternatives, quinine remained the most widely used antimalarial until the 1920s when a new breed of compounds was discovered . Drugs such as chloroquine then became the treatment of choice for malarial prophylaxis and were used widely in all areas with endemic malaria for many decades . By the 1950s, increasing levels of chloroquine resistance necessitated a push for the discovery of novel compounds for malarial chemoprophylaxis . The synthetic quinoline derivative mefloquine (bis(trifluoromethyl)-(2-piperidyl)-4-quinolinemethanol), an effective antimalarial but potent neurotoxin, was identified as part of this discovery process . First synthesised in the late 1960s, mefloquine's potent antimalarial properties were identified as part of a two - phase us military drug discovery programme that was mounted to identify novel antimalarial compounds for use primarily in their theatres of operation in southeast asia [4, 5, 710]. Studies showed that chloroquine and mefloquine acted via the same erythrocyte accumulation mechanism, but with mefloquine showing greater affinity, likely the mechanism for its increased efficacy both as a treatment and a prophylactic compared to chloroquine . Despite historical knowledge of quinine and quinoline - induced related adverse drug reactions [7, 12], including hearing loss, visual disturbances, and severe hypoglycaemia [1315], mefloquine was expeditiously developed with the assistance of the us government and the pharmaceutical company hoffmann la roche [16, 17] and released following limited clinical testing [18, 19]. Over the next twenty years mefloquine was widely advocated as the drug of choice for travellers to areas known to be endemic for chloroquine - resistant malaria such as sub - saharan africa [21, 22]. During this time well tolerated, safe, and effective despite coincident reports of significant neuropsychiatric side effects in isolated cases . During the 1990s and 2000s, an increasing body of clinical case material reported significant neuropsychiatric side effects presenting in patients taking mefloquine for malarial prophylaxis [2533]. Clinical presentation included a range of neurological symptoms in previously healthy individuals which included tremor, balance disturbances, fatigue, nausea, dizziness, anxiety or panic attacks, sleep disturbances including insomnia and vivid nightmares, visual disturbances, and hearing loss [31, 34], as well as severe neuropsychiatric sequelae including major personality change, psychosis, seizures, suicidal ideation, and suicide completion [26, 27, 3133, 35, 36]. Toxidrome, a collection of significant neurological symptoms affecting balance, vision, hearing, memory, personality, and emotional status, has now been described as a limbic encephalopathy with central vestibulopathy, an overarching diagnosis covering all the possible manifestations of this complex neurotoxicity . This review will consider how mefloquine might induce this wide range of clinical effects in the central nervous system and explores current knowledge surrounding its binding partners at the cell surface . It will also present evidence suggesting destabilisation or destruction of the brain's central pacemaker, the substantia nigra, as a unifying hypothesis underlying many of the neuropsychiatric features of mefloquine toxicity . The incidence of adverse reactions to mefloquine treatment and/or prophylaxis has long been a point of controversy . Early studies suggested that patients did not experience the very severe neuropsychiatric side effects that had been reported with chloroquine [3842] but as increasing numbers of adverse events began to be reported in the literature, this opinion changed . Recently, controlled clinical trials have suggested that the incidence of neuropsychiatric side effects in travellers using mefloquine for malarial prophylaxis as well as those for treatment of malaria was more than a hundredfold greater than had been suggested in early studies investigating drug safety [32, 4346]. However, despite significant reporting of the clinical manifestations of mefloquine toxicity, factors underlying the variability in presentation and severity of clinical signs observed in a subset of patients presenting with significant adverse reactions have yet to be fully elucidated . Some of the pharmacological properties of mefloquine, which contribute to its efficacy as an antimalarial, may also contribute to its neurotoxicity . Mefloquine has a long plasma half - life (1328 days), which contributes to its efficacy as a prophylactic treatment achievable by easy weekly dosing [47, 48]. Mefloquine is also highly lipophilic and exhibits stereoselective passage across the blood brain barrier (bbb) [4851]. In the brain, highest concentrations have been reported in the hippocampus and subcortical areas in rodent studies [52, 53] with samples from human postmortem tissues shown to be up to 10-fold higher than plasma levels [50, 54]. One mechanism likely to cause increased retention of mefloquine in the cns is via inhibition of the membrane efflux pump p - glycoprotein . P - gp (also known as atp - binding cassette protein 1, abc1), encoded by the multi - drug resistance gene 1 (mdr1), is a transmembrane protein found lining the brain capillary endothelium that plays a specific role in central neuroprotection by restricting access of lipophilic molecules across the bbb . The normal function of p - gp is to protect the brain from neurotoxic attack by limiting cns access to complex molecules; mefloquine has been shown to be a potent inhibitor of p - glycoprotein, blocking its action at the bbb and causing retention of mefloquine in nervous tissues . Effectiveness of the cyp450 enzyme superfamily in oxidative enzymatic degradation of common pharmaceuticals, including mefloquine, is likely to also play a significant role in the presentation and severity of neuropsychiatric side effects as genetic polymorphisms in the enzymes cyp2d6, cyp2c19, cyp3a4, and cyp1a2 have all been implicated in adverse reactions to common antidepressants including incidences involving significant violence and suicide . In these cases, ultrarapid metabolism was linked to suicide and extreme violence [58, 59] resulting from rapid conversion to toxic metabolites or bioactive drug production, whilst activity caused by single or multiple allelic mutation can cause failure of systemic depletion of the parent compound, with increased risk of adverse reactions to common neuropsychiatric drugs . Recently, the relationship between treatments responses and genetic polymorphism at the cyp2a6 and cyp2b6 loci were investigated in patients receiving dual artesunate - mefloquine treatment for p. falciparum malaria where mutations in cyp2a6 were related to poorer treatment outcomes due to reduced metabolic conversion of artesunate to dihydroartemisinin . Serum level of mefloquine was not measured but it might be assumed that these would also be high as low dose chloroquine has been shown recently to induce severe neuropsychiatric symptoms in an individual with mutation of the cyp2a6 and altered cyp450 activity, symptoms that were further exacerbated by treatment with other common psychiatric drugs . These findings suggest that allelic variation in cyp450 significantly increases the risk of severe neuropsychiatric sequelae on exposure to quinoline derivatives and further pharmacogenetic studies are warranted to investigate this possibility . Mutations in genes associated with cellular metabolism of toxic compounds are not the only polymorphisms implicated in increased risk of adverse neuropsychiatric events associated with quinoline therapy . Mutations at the mndr1 locus have also been shown to be associated with a heightened sensitivity to mefloquine in vitro as well as being correlated with an increased incidence of neuropsychiatric side effects in humans particularly in women . The malarial homologue of p - gp is also implicated in conferring chloroquine resistance to some strains of plasmodium falciparum [63, 64] suggesting a common role and important role for these transmembrane proteins in restricting passage of mefloquine into cells under normal condition . The highly lipophilic nature of mefloquine is also likely to be a contributory factor to the variable presentation of adverse neuropsychiatric events in humans . It has been shown that travellers with a low body weight index, taking mefloquine for malarial prophylaxis, showed an increased likelihood of adverse reactions than those taking chloroquine or the combination therapy chloroquine and proguanil . This is likely due to reduced binding of the active compound to body fat stores and therefore a preferential compartmentalisation in other lipophilic tissues, such as the brain . Certainly volume of distribution is known to play a key role in drug toxicity and it has been well documented for mefloquine's parent molecule quinine that compartmentalisation, elimination, and excretion are all affected by age and health status of the patient . In particular, rates of elimination have been found to be slower in the elderly and those suffering from acute malaria than young or well individuals [47, 65] and similar effects have been identified in patients with active malarial infection treated with mefloquine . Together, these findings suggest that variability in plasma half - life, activity of efflux pumps of the cns / vascular interface, and compartmentalisation, as well as underlying genetics regulating neuroactive drug sensitivity and metabolism, may all contribute to toxic loading of mefloquine in the cns and therefore the highly variable presentation of adverse events in a subpopulation of mefloquine users . Thus, multiple factors affecting drug retention and concentration in the cns make accurate prediction of adverse events in patients exposed to mefloquine highly problematic . It has been well described that toxic loading of mefloquine in the cns can be subject to significant interpersonal variation; however, this does not fully explain the highly varied expression of adverse side effects reported by individuals exposed to therapeutic levels of mefloquine . At toxic doses, mefloquine has been widely reported to cause neuronal dysfunction, axonal degeneration, and neuronal cell death in a variety of cell types of the central nervous system [6770], yet in some patients it appears to be able to elicit these significant and detrimental effects at much lower doses . How mefloquine induces multiple effects across a range of different neuronal subtypes is not yet clear but understanding its membrane binding partners in the cns, and effects on cell membrane excitability, could give some clues as to its varying modes of toxicity in the human brain . One family of neuronal cell membrane channels likely to play a key role in mefloquine neurotoxicity are the octomeric atp - sensitive potassium (katp) channels . Katp channels are found in a wide range of tissues, including cardiomyocytes, smooth muscle, hormone secreting cells of the pancreas, and neurons of the cns [71, 72]. Katp channels contain two components, a pore - forming kir subunit and a sulfonylurea receptor (sur) subunit, members of the atp - binding cassette superfamily [73, 74]. A number of variants of these receptor subcomponents have now been identified, with different subunit pairings found in different cell types in the body . The kir subunit forms the channel pore and contains the atp - inhibition site, whilst the sur subunit is sensitive to sulphonylureas and channel agonists [7577]. Katp channels have been shown to play a fundamental role in glucose homeostasis via their activity in insulin - secreting islet cells of the pancreas [7881] and are also widely distributed throughout the brain, in particular, being found within the cell membranes of postsynaptic -amino - butyric acid (gaba) inhibitory neurons of the cerebral cortex, substantia nigra pars reticulata (snr), pars compacta (snc), and cerebellum [73, 77, 8284]. In neurons, katp channels modulate the availability of atp for cellular metabolism by responding to depletion of atp resources within the cell . Therefore, in neurons under normal metabolic conditions, katp channels are closed, only opening when the intracellular atp / adp ratio decreases sufficiently to required metabolic homeostasis to be restored (figure 1). What is important in the context of neurotoxicity is that this modulation in atp - dependent membrane excitability confers dual properties to the cell: (1) influencing maintenance of normal spontaneous firing patterns as well as (2) conferring neuroprotective properties to the cell in instances of metabolic stress, such as in hypoxia or ischaemia [82, 85]. Katp channel blockade alters neuronal excitability and negates the neuroprotective effect, potentially leaving cells susceptible to ischaemic or excitotoxic cell death under conditions of metabolic stress or disease (figure 1). Initial clues as to a role for katp channels in mefloquine toxicity came from studies investigating metabolic abnormalities in patients treated with quinine and its derivatives . Mefloquine, chloroquine, quinoline, and quinine have all been shown to block the activity of katp channels in islet cells of the pancreas, increasing insulin secretion [86, 87]. This finding, associated with reports of severe hypoglycaemia in patients undergoing treatment with both quinine and mefloquine for malarial prophylaxis or acute disease [15, 88, 89], suggests a similar mode of action for the two compounds in the pancreas in humans . Quinine and quinoline derivatives, therefore, are likely to impede katp channel function in other tissues such as the brain and there is growing evidence to support this hypothesis . Katp are widely distributed throughout the brain and have been shown to be present in gaba - responsive neurons of the cerebral cortex, substantia nigra pars reticulata (snr), pars compacta (snc), and cerebellum [73, 82]. Of particular relevance to this review is the finding that gabaergic neurons of the snr have been shown to express katp channels, cells which are known to play a key role in maintenance of normal spontaneous firing activity in the brain [84, 90]. This spontaneous excitation pattern has been described as the fast spiking pacemaker of the central nervous system, instructing the tonic output activity of the basal ganglia and other subcortical regions, and is fundamentally required for normal neurological function . Under normal metabolic conditions, katp channels in the snr are closed and cells exhibit a high level of spontaneous activity, suppressing seizure activity by release of gaba onto postsynaptic terminals (figure 1(a)). Conversely, in states of metabolic tension, such as hypoxia, these channels are activated causing a protective hyperpolarisation due to calcium efflux, and thus reducing membrane excitability (figure 1(b)). Katp channels in the snr have been shown to play a significant role in both neuronal protection and seizure suppression [82, 92] with the majority of neurons in the snr being gabaergic and exhibiting high levels of spontaneous firing . Mefloquine inhibition of snr katp channels would open the membrane pore, initially maintaining high levels of spontaneous activity and gaba release regardless of metabolic status (figure 1(c)). Continued inhibition would result in depletion of cellular metabolic stores, reducing gaba release [93, 94], and finally cell death of snr neurons (figure 1(d)). This blockade would also confer an inability to regulate excitability in target neurons of the mesostriatal dopaminergic pathway, similar to the hyperexcitability observed in hypoglycaemia, increasing dopamine release and potentially resulting in excitotoxic cell death in target neuron populations (figure 1). Katp membrane mefloquine channel inhibition would also reduce the neuroprotective ability of midbrain neurons to respond to metabolic atp depletion making them more sensitive to metabolic stress in states or injury or disease . Mefloquine could therefore induce both neuronal dysfunction and cell death in this critical regulatory region of the brain . Another interesting and relevant finding is that extracellular dopamine levels in the striatum increases as the base firing rate of snr neurons increases . This biochemical change could potentially deregulate the delicate balance between serotonergic and dopaminergic control of the mesolimbic system (figure 2), such as what is seen in the alterations of mood and behaviour associated with states of addiction to psychostimulants and neuronal synchronisation characteristic of limbic seizures . Limbic seizures are classified as psychogenic seizure events without major epileptiform changes, which result in paroxysmal episodic alteration in cognitive function, behaviour, and emotional control [98101]. Firstly, the snr has been identified to be the site of action of the anticonvulsant topiramate in the intrahippocampal pilocarpine model of limbic seizures, exerting its action by either direct connection to the hippocampus or indirect subcortical connections via the striatum (figure 2) [102106]. Secondly, sustained opening of katp channels was induced by mild hypoxia in mice without a functional kir6.2 subunit, causing neuronal depolarisation and enhanced membrane sensitivity, sufficient to cause excitotoxic cell death [82, 85]. Seizure activity induced by mefloquine antagonism resulting from loss or dysregulation of snr tonic firing could therefore give rise to a number of the side effects observed in clinical cases of mefloquine toxicity, including significant neuropsychiatric disturbances . Effects of mefloquine exposure to cells of the snr has been examined in some detail in vitro . Mefloquine has been shown to cause hyperexcitation in primary dopaminergic neurons of the snr, increasing pacemaker firing activity in a concentration dependent manner . Significantly, this effect was observed at concentrations far lower than those found in the plasma of patients treated with mefloquine for malarial prophylaxis (0.310 mm compared to 3.823 mm in humans). This study also showed that this increased firing pattern enhanced gabaa - receptor mediated synaptic transmission by increasing intracellular calcium and inhibition of cholinesterase . In a whole animal system, this could result in sustained binding of endogenous acetylcholine to its receptors, potentially causing some of the adverse neurobehavioural and cognitive effects reported in patients undergoing mefloquine treatment . Whilst this hypothesis does not account for all of the prodromal and acute neuropsychiatric symptoms associated with exposure to mefloquine, and further work is needed to evaluate the role of cholinergic stimulation in mefloquine toxicosis, it is compatible with a past description of mefloquine toxicity as a central anticholinergic syndrome as well as known correlations between anticholinergic medications and impaired cognitive and motor function [108, 109]. Together these studies suggest that mefloquine blockade of katp channels in the snr could manifest as many of the abnormal behaviours, including heightened states of anxiety, aggression, antisocial or criminal behaviour, or seizures, widely associated with mefloquine toxicity, a hypothesis that could be investigated further in human patients experiencing adverse reactions to mefloquine prophylaxis or treatment [36, 37, 110, 111]. Further evidence exists to support a hypothesis of a role for katp channels, and their blockade, in clinical presentation of movement, auditory, and visual disturbances associated with mefloquine toxicity . Neurons of the snr interact with subcortical areas directly via the striato - nigral pathway and indirectly through the striato - pallido - subthalamic - nigral pathway, the former exerting a strong gaba - mediated inhibitory role on more posterior brain regions, including the cerebellum via connections in the pedunculopontine tegmental nucleus (figure 2) [90, 112]. Gabaergic neurons of the striatum express katp channels on their terminal axons, as well as postsynaptic channels being present on snr neurons themselves . In the nigropedunculopontine pathway, snr neurons extend direct connections to pontine cerebellar structures, the superior colliculus, and the pedunculopontine tegmental nucleus, where they play a role in modulation of saccadic and pursuit eye movements in response to sensory and attention signals from the cortex, as well as balance and coordination . A dysregulation of inhibitory input to visual or auditory centres could therefore account for auditory and visual hallucinations frequently reported in cases of mefloquine psychosis [37, 114117]. A link between neuronal activity in the snr and katp mefloquine blockade may also underlie some of the variation in presentation of adverse effects noted in travellers and patients using mefloquine for malarial prophylaxis or treatment . The snr is a region of the brain slow to reach functional maturity and shows sex specific differences in its activity . It has been reported that children tolerate mefloquine treatment better than adults and male better than female patients . These functional differences in the snr between juveniles, adults, and the elderly, as well as between men and women, could account for some of the significant variation observed in adverse effects of mefloquine treatment and prophylaxis . Genetic variance or mutation in the katp channel subunit genes kir6.2 and sur1 could underlie some of the interpersonal variation observed in patients suffering from adverse effects of mefloquine exposure . Genetic variation in kir6.2 and sur1 subunits have been shown to cause both type 1 and type 2 diabetes, as well as epilepsy in humans [121125]. As such, genetic variation in these genes might therefore predispose some individuals to more severe adverse events when taking quinoline derivatives, such as mefloquine, for malarial prophylaxis or treatment . Genetic screening for sequence variations in the kir6.2 and sur1 subunits in patients presenting with severe neuropsychiatric symptoms after mefloquine exposure could begin to define these connections more definitively and provide a better understanding as to the origin of the clinical variation presenting in cases of mefloquine toxicity . The connection between mefloquine disruptions of interneuronal communication via blockade of connexins channels, a family of gap junction family proteins, is now well established . Like katp channels, connexins (cx) play an important role in neuronal metabolism and homeostasis by controlling movement of ions, metabolites, and other molecules between adjacent cells of the cns . Gap junctions establish electrical coupling by allowing intercellular exchange of ions and metabolic support by transport of adp, glucose, glutamate, and glutathione, as well as movement of second messengers such as cyclic amp . A wide variety of pharmacological agents have been shown to influence their activity and dysfunction of connexin channel activity, or their blockade, has been implicated in a number of neuropsychiatric disorders common with mefloquine toxicity including suicide completion, vestibular dysfunction, and epilepsy . There is a known functional connection between connexins and katp channels in states of neuropsychiatric abnormality . It has been shown that katp channels regulate the expression of cx43 and cx45 in the epileptic hippocampus . Cx36 is widely expressed in cortical, subcortical, and limbic regions of the brain and has been implicated as a player in mefloquine toxicosis [132, 133]. Interestingly, cx36 is also expressed by dopaminergic neurons of the snr and gabaergic neurons of the ventral tegumental area [134, 135], again linking activity of connexins to those membrane effects of katp channels in these regions . Both mefloquine and quinine have been shown to selectively block the activity of cx36 and cx50 channels in vitro, mefloquine with significantly higher potency than its parent molecule [126, 136, 137] and mefloquine inhibition of cx36 in the inferior olive in humans has been shown to diminish motor learning skills . A connection between mefloquine, connexins, and altered neurosensory function has also been identified . Mefloquine inhibits cx26, dominant mutations of which have been shown to cause neurosensory deafness as well as attenuating increased membrane currents in primary cells expressing a dominant negative human cx26 channel . The possibility of dual dysfunction of both katp and connexin membrane channels (figures 1(c) and 1(d)), giving rise to both intra- and intercellular changes in neuronal metabolism and activity, could therefore underlie some of the very severe neuropsychiatric events observed in cases of mefloquine toxicity . Together, these studies suggest a synergistic role for connexins regulating intercellular excitability, and katp channels regulating intracellular metabolism, in the pathogenesis of mefloquine toxicity via multiplex membrane channel blockade . Variation in the sensitivity of either or both membrane channels, due to age, genetic variation, interaction with other gap junction blockers, or channel antagonists, could explain the extreme variation in neuropsychiatric symptoms presenting in patients exposed to supposedly significant evidence now exists for a primary role for membrane channel blockade in the presentation and severity of adverse neuropsychiatric reactions in patients exposed to mefloquine at normal prophylactic or treatment levels . How these complex cellular interactions manifest as neuroelectrophysiological and neurochemical changes, synaptic dysfunction, or neuronal cell death is still not clear but it seems likely that the delicate balance between excitation and inhibition caused by mefloquine exposure, both intra- and intercellularly, is likely to play a central role with connexins and katp channels both implicated in this process . A diagrammatic representation of the complex interrelationship between risk and severity of adverse events, pharmacogenetics, comorbidity with other neuropsychiatric disorders, and dose / length of exposure is illustrated in figure 3 . Reduced activity or blockade of both of these membrane channels in the brain's central pacemaker and the substantia nigra pars reticulata, as well as increased sensitivity to mefloquine due to underlying allelic variation, could provide an overarching hypothesis bringing together many of the diverse neuropsychiatric events reported in cases of mefloquine toxicoses . Further studies, including functional and structural imaging of deep brain regions in patients suffering from mefloquine toxicity and examination of electrophysiological changes in cells of the substantia with mutation or variation in both katp and connexin channels on exposure to mefloquine, could begin to elucidate the delicate interplay between excitation and inhibition in cases of mefloquine toxicoses . A hypothesis of dysfunction of the central pacemaker, giving rise to mesolimbic dysregulation, could also provide novel treatment options for patients suffering from adverse reactions to mefloquine exposure in the future.
In many protocols used to administer gonadotropin, the bioactivity of luteinizing hormone (lh) has not been considered in terms of the effectiveness of follicle - stimulating hormone (fsh) alone in supporting follicular development . In clinical practice, researchers observed that many patients show hypo responsiveness to fsh, in which follicles develop slowly, and estradiol level is low; therefore, the quality and fertility of oocytes are decreased, posing a great risk of miscarriage . In early follicular stage, fsh is important in follicle recruitment; lh may also participate in folliculogenesis and induction of ovulation . Follicular stimulation protocols have been designed to induce fsh and lh to mimic the physiology of normal human folliculogenesis (1 - 3). Studies have developed protocols for controlled ovarian stimulation (cos) by using low - dose human menopausal gonadotropin (hmg) based on fsh in late follicular phase . These studies have shown that the ovarian follicle requires a minimal amount of lh activity for steroidogenesis; as a result, the sensitivity of ovaries to fsh is increased, the dosage of fsh, and the time of ovary stimulation is reduced (4 - 7). Especially for patients with advanced reproductive age, low ovary reaction and required high gonadotropin dose, the supplement of lh activity other trials have shown that exogenous lh is also essential for patients with iatrogenic lh deficiency with gnrh against down regulation (7). Decreased lh concentrations have been associated with low pregnancy rate and increased miscarriage rates in gnrh antagonist protocol (10, 11). The duration of treatment and per cycle gonadotropin dose can be reduced by adding hmg stimulation (12, 13). Exogenesis lh can be supplied with urine hmg, low - dose human chorionic gonadotropin (hcg), and recombinant luteinizing hormone (rlh). Among these factors, rlh is limited to some extent despite its high effectiveness in treating endogenous lh deficiency because of high costs . Therefore, low - dose hcg can stimulate and selectively modulate ovarian follicle function and growth . Studies have shown that hcg supplementation in the mid - follicular phase yields favorable pregnancy results in low responders (14). However, the clinical application of hcg is influenced in terms of its long half - life . Hmg can also stimulate follicular development to induce fsh activity; furthermore, this substance can be used as lh supplement to induce lh activity . In addition, hmg is cost effective; as such, doctors and patients prefer this supplement . In the present study, the present study was designed to understand the effect of hmg on the total consumption of fsh, oocyte yield, fertilization rate, pregnancy, and implantation rate in gnrha long - protocol treatment . The zona pellucida birefringence (zpb) and zona pellucida thickness (zpt) of fertilized oocytes were observed non - invasively to explore the effect of hmg on the development of zona pellucida . Oral consent of examine zona pellucida was obtained from participants who included all patients in our center from january 2007 to february 2009 . The protocol and design of this study were approved by the ethics committee of the people s hospital of guangxi . In this retrospective study, 150 cycles were retrospectively analyzed from tubal infertile women who underwent ivf - et treatment at the people s hospital of guangxi from january 2007 to february 2009 . A long conventional protocol was used, in which a subcutaneous dose of 0.1 mg / d of gnrh - agonist (diphereline; ipsen; france) plus r - fsh (merk serono; ferring, germany) was administered with or without hmg (humegon; 75 iu / ml; organon laboratories, saint - denis, france). Individual variation in the consumption of fsh was determined according to the basal reproductive hormone level and basal antral follicle count . Approximately 5000/10000 iu hcg (based on the number of follicles with diameter 14 mm and e2 level) was administered to trigger ovulation when more than two dominant follicles reached the diameter of 18 mm . The oocytes were retrieved 36 hr after hcg by transvaginal ultrasound - guided aspiration using a single lumen ovum aspiration needle (cook, australia). In gamete buffer (gb medium; cook, australia), cumulus oocyte complexes were collected under a stereomicroscope (smz - olympus, japan). We removed granulosa cells and sperms by repeatedly aspirating these cells with a needle of 135 mm in diameter, 6 hr after fertilization . The ova were then visualized under an inverted microscope with hoffman interference optics and objective to 20, 30, and 40; the stage was heated to 37c by using a temperature controlling system (minitub ht300; tiefenbach, germany). Considering the presence of the first / second polar body and germinal vesicle (gv), we classified the oocytes into three stages: mature oocytes (metaphase ii); immature oocytes (metaphase i); and gv . Observation of zpb and zpt zpb (figure 1) and zpt (figure 2) were detected using a previously described method (15). Denude oocytes were transferred separately into 3 - 5 l pre - warmed droplets of gb medium (cook) overlaid with equilibrated mineral oil (sage, trumbull, ct, usa) in glass - bottomed dishes . The zp was imaged separately using an olympus ix71 microscope with the octax polar - aide system (octax) with an oocyte equator, in which the oolemmas were clear . The obtained oocyte images were then examined to analyze the zpt along the radius direction . These data, along with the zpb score, were saved for the subsequent quantitative analysis of zpb intensity and zpt . Our study excluded mi and gv oocytes because hmg could affect oocyte maturation, which is closely related to zpb and zpt . Therefore, only mii oocytes (fertilized and unfertilized) with a polar body were observed 6 hr after insemination . Evaluation of pronuclei and embryo quality pronuclei were evaluated using system in which the presence and number of pronuclei were obtained 18 - 20 hr after insemination . The embryos were scored according to the degree of cytoplasm fragmentation and the number of blastomeres . Grade 1 embryos were considered the most viable embryo exhibiting the following characteristics: 8 - 10 cells; symmetrical blastomeres; few fragments; absent multinuclei; and rough, clear cytoplasm . Grade 2 embryos contained more than seven cells with uniform cell division and maximum 20% of cytoplasm fragment . Grade 3 embryos contained more than four cells with uneven cell division and certain proportion of fragments (> 20%, <50%). Grade 4 embryos contained poorly viable (morphologically lowest) embryos with uneven cell division and> 50% of fragments . Sperm preparation and fertilization sperm cells were harvested by sperm - grade double - density centrifugation (40% and 80%) technique (cook, australia) after these cells were incubated in ivf medium at 37c with 6% co2 for 1 - 2 hr . Oocytes were then fertilized with a progressive motile sperm with a density of 100,000 - 200,000/ml . Embryo transplantation for transplantation, embryo was selected on the basis of traditional morphological assessment depending on the third day or the fifth day of embryo development . We transplanted two high - quality embryos to patients younger than 35 years old and three high - quality embryos to those in advanced age or subjected to repeated ivf cycles . Embryos were incubated in a petri dish (falcon 3037, france) containing blastocyst medium (bm, cook, and australia) for 0.5 - 2.0 hr; these embryos were then transplanted into the uterus using an ivf transfer catheter (cook, australia) by ultrasound . In the luteal phase, progesterone was administered on the first day of transplantation and continued until the day of the pregnancy test . Pregnancy evaluation clinical pregnancy is determined on the basis of the presence of gestational sac, as confirmed by ultrasound on the fourth week after the embryo is transplanted . Statistical analysis statistical analysis was performed by anova and tests using graphpad prism 4 . Among the 150 cycles subjected to the long protocol with gnrh agonist, 81 received r - fsh treatment . A total of 69 cycles were subjected to superovulation by using r - fsh plus hmg as exogenous lh administered in the late follicular development . The average age of female patients in the two groups was comparable (31.250.41 vs. 31.330.42 years, respectively; p=0.99). No statistical difference was observed between the two groups in terms of the average number of retrieved oocytes and the maturation rate (13.90.73 vs. 11.990.75, p=0.06; 83.32% vs. 84.76%, p=0.42). The average amount of fsh in the r - fsh+ hmg group was similar to that in the r - fsh group (158360.47 vs. 159149.43, p=0.14; table i). Further studies were preformed regarding fertilization, cleavage, and embryo development after superovulation was performed with or without hmg . The results in table ii suggested that the fertilization rate was higher in the hmg group (82.95% vs. 78.75%; p=0.02). However, no difference was observed between cleavage and excellent embryo rates (94.90% vs. 94.76%, p=0.99; 57.91% vs. 57.29%, p=0.85, respectively) between the two groups . The endometrial thickness and average number of transplanted embryos were comparable between r - fsh group and r - fsh+ hmg group (11.720.31 vs. 11.180.26, p=0.19; 2.200.05 vs. 2.240.05, p=0.59; respectively). The results also indicated that pregnancy rate, implantation rate, and miscarriage rate did not differ between the two groups (37.66% vs. 37.31%, p=0.96; 23.26% vs. 28.97%, p=0.30; 17.24% vs. 8.00%, p=0.54; respectively). Zpb was evaluated 6 hr after fertilization was performed using the long protocol cycles of ivf in the two groups (table iv). No difference was found between r - fsh group (7.040.31) and r - fsh+ hmg group (6.700.50) (p=0.53). However, d0zpt and d1zpt of r - fsh group were respectively lower than those of r - fsh+ hmg group (18.750.10 vs. 19.200.14, p=0.01; 18.170.14 vs. 18.690.12, p=0.00) when the zpt of the fertilized oocytes were further analyzed at 6 or 20 hr after insemination on d0 or d1 . Comparison of the number of retrieved oocytes and oocyte maturation rate between r - fsh and r - fsh+ hmg groups in ivf cycles r - fsh: recombinant follicle - stimulating hormone, hmg: human menopausal gonadotropin data are presented as the meanse comparison of fertilization between r - fsh and r - fsh+ hmg groups in ivf cycles comparison of clinical outcomes between r - fsh and r - fsh + hmg groups in ivf cycles comparison of birefringence intensity and thickness of zona pellucida between r - fsh and r - fsh + hmg groups in ivf cycles data are presented as the mean se examination of the zona pellucida birefringence scores of mii oocytes examination of the zona pellucida thickness all of the 150 tubal infertile patients undergoing ivf treatment received a standard long protocol in this research . The average amount of r - fsh, endometrial thickness, number of retrieved oocytes, and mature rate in the r - fsh+ hmg group were not different from those in the r - fsh group; this result is similar to those in the previous reports (5, 6). No evident differences were also found in cleavage, clinical pregnancy, and implantation rates between the two groups . This finding may be attributed to the change in zona pellucida, although gordon et al found that residual endogenous lh after down regulation is sufficient for normal ovarian response and estradiol synthesis and implantation is possibly higher when lh is co - administered (6). Our study demonstrated that the zpb of oocytes was lower in the hmg+ r - fsh group than in the r - fsh group . This could be related to the higher lh level of patients treated with hmg supplement . An appropriate concentration of lh can possibly promote oocyte maturation and developmental potential (6). In addition, a previous study regarding the zona pellucida showed that the zpb of oocytes reduced gradually from a gv stage to a mii stage in the developmental process (16). These results could be possibly attributed to the changes in paracrine in ovarian follicle caused by lh, such as camp, epidermal growth factor receptor (egfr), and c - type natriuretic peptide (cnp)/ natriuretic peptide receptor 2 (npr2) (17, 18). In meta - analysis, a high serum estradiol level was obtained when hcg was administered and a high number of metaphase ii oocytes were retrieved in the hmg group when the gonadotropin releasing hormone antagonist (gnrh - a) protocol was used; however, no difference was observed in the rates of clinical pregnancy, implantation, and miscarriage (19). Lh can also improve clinical pregnancy and implantation rates in low- and normal - responsive patients (6, 7, 20). Lh is necessary to stimulate folliculogenesis and maturation or development potential of oocytes in clinical and experimental aspects (7, 21). Loumaye et al reported that lh levels increase when exogenous lh is administered; this increase may produce medium - sized follicles to atresia, even if large amounts of fsh are used in superovulation; as a result, multiple follicle development was avoided (22). In the current study, a moderate reduction in the r - fsh+ hmg group was observed compared with the control group (11.99 0.75 vs. 13.90.73, respectively), which can be closely attributed to the efficacy of hmg in the controlled superovulation, although the overall number of retrieved oocytes represented no significant difference between the two groups (p=0.06). Fabregues et al showed a remarkable decrease in the number of non - dominant follicle with a diameter of 14 - 18 mm when hcg was administered (23). The number of produced and fertilized oocytes significantly decreased in the fsh group rather than in the fsh + lh group . Previous studies revealed that exogenous lh activity (including hmg or r - lh) added in the mid - follicular phase of superovulation can optimize the controlled ovarian stimulation and ovulation outcome particularly in the subgroup of patients with advanced age and poor ovarian response (24, 25). The excessive secretion of lh can induce the release of androgen in theca cells, and this follicular microenvironment can induce not only the apoptosis of granulosa cells but also follicular atresia (26). A series of morphological changes in follicular granulosa cells and oocytes were observed during follicular atresia and degradation . Excessive lh can also negatively affect oocyte maturation; as a result, overly mature oocytes are produced and meiosis and mitosis are accelerated . In addition, granulose cells are luteinized and oocyte quality is reduced because of a premature and excessive secretion of lh before ovulation occurs; by contrast, low lh levels can produce e2 at concentrations less than normal values (7). Westergaard et al and weghofer et al reported that lh level <0.5 u / l in the mid - follicular phase likely increases the probability of abortion because of embryo polyploidy, although follicle growth, fertilization, and pregnancy remain unaffected (27, 28). This result showed that lh at an appropriate range could maintain normal levels of follicular androgen and improve follicular development and oocyte quality . Likely results in an insufficient synthesis of e2, whereas lh that exceeds the ceiling level is possibly detrimental to follicular development (29). In conclusion, hmg supplement did not elicit evident effects on normal - responsive patients except fertilization and zpb and zpt of oocytes.
The hereditary ataxias are a clinically and genetically heterogeneous group of inherited neurodegenerative disorders with ataxia as the predominant symptom . The hereditary ataxias include the autosomal recessive ataxias, the dominantly inherited spinocerebellar ataxias (sca) and episodic ataxias (ea) and the x - linked ataxias . Although the causative mutations have been identified in a number of ataxia subtypes, the underlying genetic defects for many forms of hereditary ataxia remain unknown . The transient receptor potential (trp) channel family member trpc3 is a non - selective cation channel, which is highly enriched in the purkinje cells of the cerebellum [2, 8]. Trpc3 is a key mediator of metabotropic glutamate receptor (mglur)-dependent synaptic transmission in the purkinje cells and has also been implicated in cerebellar development [2, 10]. Interestingly, trpc3 is associated with several proteins that are linked to human cerebellar ataxia, including mglur1, the ip3 receptor and protein kinase c (pkc) [8, 14]. In vitro studies have shown that trpc3 is a substrate of pkc, and that mutants of pkc-causing sca14 fail to phosphorylate trpc3, resulting in dysfunctional calcium homeostasis . Furthermore, in a mouse model of sca1, trpc3 is downregulated before the onset of degeneration . Notably, genetic mouse models of trpc3 dysfunction exhibit cerebellar ataxia [2, 8]. Collectively, these studies point to an important role of trpc3 in the pathways leading to cerebellar ataxia in mice and humans and suggest trpc3 as a promising candidate gene for hereditary forms of human cerebellar ataxia . Here, we report a mutation screen of the human trpc3 gene in a cohort of 108 patients with suspected but, as yet, undetermined genetic forms of ataxia . Ninety - eight samples were from patients with late - onset cerebellar ataxia seen at a large tertiary referral centre . Patients had sporadic onset or did not have a clearly defined family history of ataxia . Evaluation was performed for acquired and hereditary causes of late - onset cerebellar ataxia according to established clinical protocols, and patients did not have a defined genetic diagnosis at the time of screening . Prior genetic analysis was performed individually based on phenotype but, in general, minimal analysis included screening for sca1, sca2, sca3, sca6, sca7 and fxn repeat expansions . Patient consent was obtained and dna was extracted from peripheral blood leukocytes using the gentra puregene blood kit (qiagen). All human subject research protocols were approved by the ucla institutional review board and comply with the current laws of the united states . Dna samples from ten individuals with clinical features consistent with episodic ataxia type 2 (ea2) were obtained with informed consent . These patients had been previously sequenced for mutations in the coding and flanking intronic regions of cacna1a and no disease - causing mutations were identified . Dna from 96 healthy adults (european collection of cell cultures) were screened as controls . Patient genomic dna extracted from blood was amplified using standard pcr techniques with primers for all coding regions of trpc3 (exons 111 of the trpc3 - 001 transcript according to the ensembl genome browser 58) and an average of 80 nucleotides of flanking intronic regions . All amplicons were sequenced using big dye 3.1 dideoxy terminator methods (applied biosystems) using the amplification primers . To confirm sequence variants, exonic splicing enhancer (ese) motifs were predicted using esefinder 3.0 (cold spring harbor laboratory) and rescue - ese . Ninety - eight samples were from patients with late - onset cerebellar ataxia seen at a large tertiary referral centre . Patients had sporadic onset or did not have a clearly defined family history of ataxia . Evaluation was performed for acquired and hereditary causes of late - onset cerebellar ataxia according to established clinical protocols, and patients did not have a defined genetic diagnosis at the time of screening . Prior genetic analysis was performed individually based on phenotype but, in general, minimal analysis included screening for sca1, sca2, sca3, sca6, sca7 and fxn repeat expansions . Patient consent was obtained and dna was extracted from peripheral blood leukocytes using the gentra puregene blood kit (qiagen). All human subject research protocols were approved by the ucla institutional review board and comply with the current laws of the united states . Dna samples from ten individuals with clinical features consistent with episodic ataxia type 2 (ea2) were obtained with informed consent . These patients had been previously sequenced for mutations in the coding and flanking intronic regions of cacna1a and no disease - causing mutations were identified . Dna from 96 healthy adults (european collection of cell cultures) were screened as controls . Patient genomic dna extracted from blood was amplified using standard pcr techniques with primers for all coding regions of trpc3 (exons 111 of the trpc3 - 001 transcript according to the ensembl genome browser 58) and an average of 80 nucleotides of flanking intronic regions . All amplicons were sequenced using big dye 3.1 dideoxy terminator methods (applied biosystems) using the amplification primers . To confirm sequence variants, exonic splicing enhancer (ese) motifs were predicted using esefinder 3.0 (cold spring harbor laboratory) and rescue - ese . To identify possible mutations in trpc3, we screened all 11 coding exons and flanking exon / intron boundaries of this gene in 98 patients with undiagnosed late - onset cerebellar ataxia . Previous work in mice had suggested that trpc3 mutations cause a dominant pure cerebellar phenotype, therefore patients with similar phenotypes were selected from a population of undiagnosed ataxia cases at a large tertiary referral centre . Late - onset cases (onset greater than age 25 years) were chosen as this is the most common presentation of a dominantly inherited cerebellar ataxia . Table 1characteristics of the patient populations studiednumberage at exam (years)female (%) cerebellar only (%) sca - like (%) spasticity (%) episodic (%) msa - like (%) 9855.5 15.857.128.637.812.28.213.31025 1433.30001000ninety - eight patients presented with late - onset gait and appendicular ataxia . Additional phenotypic features include involvement of spinocerebellar pathways and/or peripheral neuropathy (sca - like), increased lower and/or upper extremity tone (spasticity), fluctuating periods of ataxia (episodic) or autonomic neuropathy and/or parkinsonism not diagnostic for multiple system atrophy (msa - like). A further panel of ten patients presented with episodic ataxia clinically similar to ea2 characteristics of the patient populations studied ninety - eight patients presented with late - onset gait and appendicular ataxia . Additional phenotypic features include involvement of spinocerebellar pathways and/or peripheral neuropathy (sca - like), increased lower and/or upper extremity tone (spasticity), fluctuating periods of ataxia (episodic) or autonomic neuropathy and/or parkinsonism not diagnostic for multiple system atrophy (msa - like). A further panel of ten patients presented with episodic ataxia clinically similar to ea2 as trpc3 is an ion channel, it is possible that the human phenotype may demonstrate a paroxysmal course . We included an additional ten samples from patients with a dominant history of episodic ataxia whose clinical features were most consistent with ea2 in terms of attack precipitants and duration, interictal signs and response to acetazolamide . All ten individuals were initially sequenced for mutations in the cacna1a gene and found to be negative . Three known single nucleotide polymorphisms (snps) were found in the patient cohort at similar frequencies as published for the hapmap - ceu control population (table 2). In addition, we identified two novel variants (c.585g> a, c.2271a> g), each in a single patient (table 2). Both of these snps result in synonymous changes at the protein level (p.lys195lys, p.ser757ser). Interestingly, the c.2271a> g variant in exon 9, which was found in a patient with a complex phenotype of intermittent ataxia and episodic hemiplegia, is predicted to weaken an ese motif according to bioinformatics calculation . The mutation was also detected in the proband s mother, who had hemiplegic migraine . In an in vitro splicing assay, this putative ese motif did not have an effect on the splicing of an artificial minigene construct (data not shown). However, this does not rule out the potential importance of this ese motif for splicing of the full trpc3 gene in vivo in the patient s nervous system . Unfortunately, this patient was lost to follow - up and material was not available for further splicing analysis . Table 2identified genetic variants in trpc3refsnpvariationexonaa changegenotype frequencygenotype frequency in populationrs13121031c.78c> g1p.ala26ala0.148 (g / c)0.183 (g / c)c.585g> a1p.lys195lys0.009 (c / t)n / ars11732666c.2199g> a8p.arg733arg0.352 (c / t)0.5 (c / t)0.111 (t / t)0.1 (t / t)c.2271a> g9p.ser757ser0.009 (t / c)n / ars61741700c.2451a> g10p.glu817glu0.019 (c / t)n / anomenclature is based on the national center for biotechnology information (ncbi) reference sequences nm_003305.2 (mrna) and np_003296.1 (protein). Snp reference numbers, genetic variants and population frequency (hapmap - ceu) are listed according to the ncbi dbsnp database . Newly identified variants are indicated in boldn / a not applicable identified genetic variants in trpc3 nomenclature is based on the national center for biotechnology information (ncbi) reference sequences nm_003305.2 (mrna) and np_003296.1 (protein). Snp reference numbers, genetic variants and population frequency (hapmap - ceu) are listed according to the ncbi dbsnp database . Genetic mouse models of loss of trpc3 or dysfunctional trpc3 result in an ataxic phenotype [2, 8]. Furthermore, several studies have linked trpc3 to key signalling pathways that are affected in human cerebellar ataxia [1, 14]. Trp family member genes have recently been implicated in human neurological diseases [4, 5]. We, therefore, hypothesized that mutations in the trpc3 gene might underlie previously undiagnosed late - onset forms of human cerebellar ataxia or episodic ataxia . Our findings suggest that mutations in trpc3 are not a common cause of late - onset or episodic cerebellar ataxia . We did identify a novel variant in one patient with episodic ataxia that is predicted to alter splicing of the trpc3 gene . Protein interaction motifs in the intracellular c - terminus, including the crib and cam binding domains . As a consequence, this shorter form of trpc3 could act as a dominant negative and impair proper calcium handling in purkinje cells of the cerebellum . Despite the absence of trpc3 mutations in our group of patients, trpc3 mutations may still contribute to human disease, as it is well - known that genetic mutations do not always produce equivalent phenotypes in mice and humans . Therefore, future genetic studies in larger patient cohorts and/or different ethnic / geographic populations, as well as in patients with different subtypes of cerebellar ataxia, are necessary to fully elucidate the role trpc3 may play in human cerebellar ataxia.
Pathogens have evolved ingenious strategies to circumvent the host immune response as part of the constant evolutionary process - taking place in all living organisms . Chief among these strategies is the prevention of the inflammatory response or seizure of the anti - inflammatory mechanism in place to protect tissue integrity . The manipulation of macrophage (m) polarization is one of the main targets to accomplish this, since this antigen presenting cell represents the first line of an active defense system in the host, and if successfully done, it can then undermine adaptive immunity (benoit et al ., 2008). M polarization is a dynamic process governed by mechanisms dictating their tissue distribution and functional capacities in response to endogenous and exogenous signals (martinez et al ., 2009). Polarized ms are broadly classified into two groups: classical (m1) and alternative (m2) activated . On one hand, m1 program is a direct response to type-1 inflammatory conditions (e.g., ifn-) and pathogen challenge, and it has been associated to resistance to intracellular pathogens and to some form of tumors . On the other hand, the m2 program is driven by type-2 inflammatory signals such as il-4 and il-13 (m2a); immune complexes, toll - like receptors (tlrs) agonists, or il-1 receptors (m2b); and immunosuppressants including il-10, transforming growth factor- (tgf-) or glucocorticoids (m2c; table 1). M2 ms participate in diverse activities including the suppression of inflammation, enhancement of phagocytosis, promotion of tissue remodeling and repair, elimination of parasites, and unwanted tumor angiogenesis (sica et al ., 2008; martinez et al . Furthermore, it is becoming clear that m polarization supports different, and in some cases, opposing biological functions, that influences tissue homeostasis, and numerous pathological situations, including infectious diseases (benoit et al . Given the pivotal role ms play as sentinels of the immune system, they represent ideal cell targets for subversion by successful intracellular pathogens . Priming stimulus for the classical (m1) and alternative (m2a - c) activation of macrophages . The purpose of this short review is not to provide a comprehensive summary of m polarization; others have recently reviewed this growing research area (martinez et al . Also, we will not address the multiple ways by which the pathogens in question circumvent the immune system, as there are excellent reviews covering this subject (deretic et al ., 2004; carter and ehrlich, 2008; meena and rajni, 2010; hajishengallis and lambris, 2011). Instead, we will focus exclusively on the significance of m polarization in the context of pathophysiology caused by mycobacterium tuberculosis (mtb) and human immunodeficiency virus (hiv). The world health organization reports tuberculosis (tb) is still one of the leading causes of death due to a single infectious agent (mtb) with 1.7 million deaths and 9.4 million new cases in 2009, and estimates that about one - third of the human population may be latently infected (who global tuberculosis control report 2010, 2010). Active tb may occur directly after infection or through the reactivation of latent infection that is confined in granulomas . The elaboration and maintenance of granulomas depends on a dedicated immune response, which is not fully understood . Recently, however, it was demonstrated mycobacteria exploits m activation to turn the granuloma into an effective tool for pathogenesis (davis and ramakrishnan, 2009; volkman et al ., 2010). Therefore, a better understanding of m polarization during mtb infection might yield further clues about how mtb circumvents the immune system . As aforementioned, m polarization is mainly driven by type-1 and type-2 inflammatory signals (table 1). Type-1 inflammatory cytokines are essential in the defense against mtb since their expression often correlates with efficient anti - mtb immune responses, and genetic deficiencies of these factors lead to increased tb susceptibility (quintana - murci et al ., 2007). Ifn- drives the m1 program characterized by m capacity to kill most mycobacteria and restrict the replication of the remainder (ehrt et al ., 2001). The early phase of the anti - mtb immune response is marked by m1 m polarization in multiple animal models and reminiscent of the clinical data collected from patients with active tb (figure 1; benoit et al ., 2008). At the transcriptome level, the gene modulation induced by mtb in ms highly overlaps, and in some cases synergizes, with that induced by ifn- to establish the m1 phenotype (ehrt et al . M1 m polarization is evident in mice between 7 and 30 days after mtb infection when high levels of ifn- and inos are also detected within this structure and around the alveolar compartment (redente et al ., 2010). All in all, polarization of m1 ms is part of the common host response against intracellular bacteria characterized by high expression of inos and consequent nitric oxide (no) production (characteristic of murine models), secretion of pro - inflammatory cytokines and chemokines, release of proteolytic enzymes and anti - microbial peptides, enhanced phagocytosis, and development of a toxic intracellular environment reflected in the fusion of microbial phagosomes with acidic and hydrolase - rich lysosomes (ehrt et al ., 2001; deretic et al ., 2004; martinez et al ., 2009; cairo et al ., 2011; murray and wynn, 2011). It remains to be demonstrated whether transcription factors [e.g., p65 and interferon regulatory factor (irf5)] or regulators (e.g., ship1) that dictate the m1 program of macrophage polarization also play a role in tb infection (martinez, 2011). Considering this hostile environment created by m1 ms, it is not surprising mtb has evolved strategies to interfere with m1 polarization . Indeed, mtb inhibits ifn- activation of ms by secreting virulence factors such as lipoarabinomannan that halters phagosome maturation, or early secretory antigenic target-6 (esat-6) that prevents the activation of nf-b and ifn- regulatory factors downstream of tlr-2 (deretic et al ., 2004; benoit et al ., 2008). Indirectly, mtb blocks m1 polarization by the transcriptional inhibition of ifn--responsive genes through a bystander effect involving il-6 (sibley et al ., 1990; benoit et al ., 2008). A model illustrating the putative involvement of macrophage polarization during mtb or hiv-1 infection . Perhaps the best strategy to avoid the challenges posed by m1 ms is to shift their program into m2 ms . Tb susceptibility parallels with elevated levels of type-2 inflammatory signals (e.g., il-4, il-13; kahnert et al ., 2006; raju et al ., 2008; almeida et al ., 2009; schreiber et al ., likewise, high levels of il-10 (mostly derived from ms) correlate with active tb patients (barnes et al ., 1993; verbon et al ., 1999 interestingly, the predominant type-2 inflammatory environment shifts back to type-1 after successful treatment of pulmonary tb in infected patients (verbon et al ., 1999; 2008). These observations in humans parallel with those reported in mtb - infected mice; that is, there is an early type-1 immune response characterized by ifn- during the first 3 weeks after infection, followed by a type-2 immune response that contains high levels of il-4 (figure 1; orme et al ., 1993). A type-2 inflammatory environment drives the m2 program that renders ms immunomodulatory and poorly microbicidal (raju et al ., 2008;, this seems to be the case in mice since m2 ms displayed a diminished inflammatory response to mtb as reflected by a reduced no production and increased of iron availability, suggesting these phagocytes offer a permissible intracellular environment for bacterial replication (kahnert et al ., 2006). Indeed, ifn--induced no production is essential for host survival with respect of experimental tb, while iron - starvation is key to bacteriostasis (ehrt et al ., 2001; forbes and gros, 2001; cairo et al ., 2011). It remains to be seen if mtb also influences the expression level of kruppel - like factor 4 (klf4) or any other transcription factor / regulator recently shown to be critical for both the establishment of the m2 program and the inhibition of m1 polarization (e.g., stat6, cot / tpl2; liao et al ., it has been demonstrated that both il-4 and il-13 inhibit autophagy in m1 ms resulting in enhanced survival of mtb, an impairment that might also extend to m2a ms (harris et al ., 2007). At the granuloma level in mice, inos continues to be expressed within this structure but a significant shift from m1 toward m2 ms [inos arginase-1 (arg1)] occurs around the alveolar compartment starting at day 35 and continuing up to day 60 after mtb infection, accompanied by high levels of type-2 inflammatory signals (ly et al . 2010). Given the development of fibrosis is a key characteristic of caseous granulomas during mtb dissemination, and that m2 ms have been implicated in the inhibition of fibrosis development, the shift from m1 into m2 program might represent an attempt by the host to halter the pathophysiology caused by mtb or a microbial strategy to shield from immune attack (dorhoi et al ., 2011). For instance, mtb might influence all tlr - dependent signaling by targeting dc - sign to induce il-10 and counteract the pro - inflammatory response, as shown in dendritic cells (geijtenbeek et al . Likewise, the mannosylated lipoarabinomannan from mtb enhances the production of il-10 and other immunosuppressants through recognition by the mannose receptor (mr) in immature dendritic cells (chieppa et al ., 2003). Although alveolar ms express dc - sign and mr, their role in m2 ms has yet to be demonstrated (chroneos and shepherd, 1995; tailleux et al ., 2005) (2009) reported mtb - induced il-10 in ms promotes the m2 polarization program displaying diminished anti - mycobacterial effector mechanisms . Indeed, m - specific overexpressing il-10 transgenic mice were indeed susceptible to mtb infection, displayed a specifically suppressed il-12 in infected tissues, and were characterized by lung ms with a m2 phenotype permissive to mtb infection (schreiber et al ., 2009). These observations correlate well with another study in mice where mtb was shown to promote its survival and ability to cause disease through a myd88-dependent induction of arg1 . Arg1 inhibits no production by ms by competing with inos for arginine (the common substrate), thus rendering these cells permissive to mtb infection (el kasmi et al ., 2008; hajishengallis and lambris, 2011). Taken together, these observations suggest the reprogramming toward m2 ms by il-10, and other immunosuppressants such tgf- and glucocorticoids (hernandez - pando et al ., 2006), might be yet another adaptation by mtb to survive and thrive inside of ms (figure 1). However, it should be noticed that this phenomenon might also represent a control mechanism by the host to preserve the integrity of mucosal sites as uncontrolled type-1 inflammatory responses against mtb result into lung immunopathology (hernandez - pando et al ., 2006; human immunodeficiency virus-1 is another successful intracellular pathogen responsible for a worldwide pandemic . According to 2009 estimates by the united nations, there were about 33.2 million people worldwide living with hiv-1 infection and 2.6 million individuals had been newly infected (cohen et al ., 2011). In the absence of antiviral therapy, hiv-1 infection progresses through acute and asymptomatic stages leading to the eventual failure of the host immunological functions and acquired immunodeficiency syndrome (aids). A reason is that hiv-1 targets cells from the mononuclear phagocyte lineage that drive an effective antiviral response and simultaneously serve as reservoirs of latent or productive infection (goodenow et al ., 2003). Among these cells, ms are critical to pathogenesis because they contribute to early transmission, systemic dissemination, and persistence of hiv-1 . Indeed, hiv-1 evades immune surveillance by hiding and thriving inside ms despite anti - retroviral treatment, and when infected, they persist for months displaying insensitivity to viral cytopathic effects . In addition, ms continuously secrete high level of viral particles over prolonged time periods by storing assembled virus in specialized endosomal compartments (orenstein et al ., 1988; benaroch et al ., thus, they represent powerful long - term viral reservoirs (goodenow et al ., 2003; carter and ehrlich, 2008; herbein and varin, 2010; cohen et al ., 2011). In light of recent evidence suggesting that m1 and m2 ms influence hiv-1 pathogenesis, there is a surging interest to study the viral effects in m polarization . In vitro, this m response includes production of type-1 pro - inflammatory cytokines (ifn-, il-2, il-12, tnf, il-1, il-6, il-18) and chemokines (ccl3, ccl4, mip-, mip-, rantes), increased no and respiratory burst, up - regulation of mhc - ii molecules, and down - regulation of hiv - entry receptors (e.g., cd4, ccr5, cxcr4), and endocytic receptors (e.g., cd163, cd206; swingler et al ., 1999; cassol et al ., 2009, 2010; herbein and varin, 2010). Although few studies have examined thoroughly hiv - induced polarization of ms in vivo, there is a predominance of ms displaying a m1 phenotype during the acute stage (figure 1; cassol et al ., 2010; herbein and varin, 2010; cohen et al ., 2011). Whether m1 ms are beneficial to the host during hiv-1 infection remains an open question since m functions vary according to the experimental context . For instance, in vitro infection of m1 ms in the presence of ifn- and tnf is associated with a suppression of hiv-1 replication, a sharp decrease in hiv-1 dna synthesis at 48 h, and a decrease in the accumulation of hiv-1 proteins (cassol et al ., 2009). In addition, other studies demonstrate that m1 ms inhibit viral entry, assembly, and budding, suggesting the m1 program can be beneficial to the host (cassol et al ., 2010; herbein and varin, 2010). However, it is also known that pro - inflammatory signals deriving from m1 ms favor the formation of viral reservoirs with increased transcription of hiv-1 ltr (long terminal repeat), alluding m1 ms might benefit hiv pathogenesis (cassol et al ., 2010; herbein and varin, 2010). This is supported by multiple observations that immune activation driven by ms correlates with hiv-1 pathogenesis (goodenow et al ., 2003; lamers et al ., 2009; cohen et al ., 2011 m1hiv since it displays a pro - inflammatory state with increased production of cytokines independently of tlr - pathway . The authors argue that while hiv-1 stimulates ms through a variety of signaling pathways to promote a tailored inflammation in its favor, the tlr recognition of viral replication is impaired and could serve as a viral evasion strategy . Given that prolonged pro - inflammatory m activation during chronic hiv - infection contributes, not only to a permissive environment for the formation of viral reservoirs with strong transcriptional activity, but also to disease progression and hiv - induced tissue damage, the proposed m1hiv polarized state may render ms detrimental to the host (goodenow et al ., 2003; brown et al ., 2008; lamers et al ., 2009). As hiv - disease progresses from the acute to asymptomatic stage, there is a switch from a type-1 toward a type-2 inflammatory environment (figure 1; vasilescu et al ., 2003; becker, 2004). At the transcriptional level, lymphatic tissue microarray analyses from hiv-1-infected subjects at different clinical stages revealed that each stage has a unique gene profile (li et al ., 2009). The asymptotic phase, however, down - regulates the acute phase gene profile to baseline level while it displays an increased expression of immunosuppressive genes (li et al ., 2009). Based on these immunological systemic changes, it is likely that a polarization switch occurs in ms from a m1 program during the acute phase to the m2 programs through later stages . Although there is no overwhelming evidence confirming the abundance of m2 ms in either the asymptotic or aids phase in vivo, the fact cd163 (a m2 m cell surface marker) is considered as a potential biomarker for hiv-1 disease progression may allude to the presence of m2 ms in hiv-1-infected individuals (burdo et al ., 2011; tippett et al ., similar to m1 ms, it is not known whether m2 ms benefits the host during hiv-1 infection . In vitro activation of m2a (il-4-treated) ms results in inhibition of virus replication (cassol et al ., 2009). Other studies have demonstrated that both il-4 and il-13 down - regulate viral entry receptors and hiv-1 reverse transcription in ms (cassol et al ., 2010). Furthermore, activation of m2c (il-10-treated) ms strongly inhibits reverse transcription, transcription of hiv-1 ltr and viral assembly (herbein and varin, 2010). Based on these observations, it might be tempting to conclude that m2 ms are beneficial to host immunity against hiv . However, the progression of aids is characterized by the loss of il-2 and increase of il-10 correlating with hiv viremia (brockman et al . . Moreover, the haplotypes of both il-4 and il-10 genes have been associated recently with aids progression (vasilescu et al ., therefore, the switch toward a m2 m program might simply be part of a defensive mechanism by the host to control hiv - induced tissue damage since they participate in suppression of inflammation and promotion of tissue repair (figure 1; martinez et al . Recently, a functional proteomic analysis of hiv - infected ms in the presence of regulatory t cells showed that a deviation of m1 to m2 m program is associated with neuroprotection in the case of hiv - associated neurocognitive disorders, suggesting m2 ms may curtail the m1hiv polarized activity resulting in tissue damage (huang et al ., 2010). Conversely, the switch toward the m2 m program might also occur as an evasion strategy by hiv to promote its own survival . A recent study demonstrated that hiv up - regulates both programmed cell death ligand 1 (pd - l1) and pd - l2 expression, members of the b7:cd28 family, and pd-1 ligands, in ms (porichis et al ., 2011). Given the importance of these molecules in t cell exhaustion during hiv infection, the ability of il-10 to both activate the m2c m program and induce pd - l1, and the fact that il-10 production and increased expression of pd - l1 correlate in hiv - infected patients, the authors propose the manipulation of pdl expression in ms as a strategy to evade immune responses (trabattoni et al . Whatever the true role of m2 ms in hiv infection, it is clear they influence the establishment of hiv pathogenesis, and more studies are needed to examine thoroughly hiv - induced polarization of ms in vivo . Tuberculosis is the most common opportunistic infection in aids and often used as a clinical parameter for undiagnosed aids cases (deretic et al ., 2004). While the synergy between mtb and hiv is evident at the clinical level, the mechanisms accounting for it are poorly understood . 2004) proposed the interference with endosomal sorting machine as a molecular mechanism contributing to the synergy between these two pathogens . Likewise, we envision the pathogenic modulation of m polarization as a cellular mechanism that might influence this synergism . As aforementioned, it is estimated that about one - third of the human population may be latently infected with mtb (who global tuberculosis control report 2010, 2010), suggesting that one in three of the 2.6 million people newly infected with hiv-1 in 2009 (cohen et al ., 2011) latent mtb is confined in solid granulomas composed of mainly by ms and t cells that maintain their stability . The coinfection with hiv-1 results in a dramatic increase in the odds of latently infected people progressing into overt tb to a staggering annual risk of 10% (deretic et al ., 2004; swaminathan et al ., hiv - driven immune perturbation, reflected in the loss of cd4 t cells and abnormal low levels of tnf causes the loss of granuloma integrity and efficiency in anti - microbial containment leading to post - primary reactivation state (paige and bishai, 2010). These events may increase both m necrosis and release of intracellular bacilli accounting for the extrapulmonary tb manifestation diagnosed in patients with hiv - driven immunosuppression (swaminathan et al . The awakened mtb then might induce m1 ms to drive an excessive tnf response (together with other mechanisms such as mmp secretion) to deliberately promote parasitic granuloma formation, resulting in the recruitment of additional nave ms and the tissue pathology (davis and ramakrishnan, 2009; paige and bishai, 2010; volkman et al ., 2010). Excessive levels of tnf, may not only contribute to the classical symptoms of cachexia in tb, but also to the augmentation of hiv-1 transcription and accelerated formation of viral reservoirs (deretic et al . In the absence of an efficient adaptive immune response due to hiv - driven impairment, uncontrolled inflammation can result in lung immunopathology, and consequently, the host may induce tissue repair responses . The shift from m1 to m2 m program may become pronounced and prolonged in the sterile attempt to restore tissue integrity, elevating the level of il-10 that is typical of disease progression by both pathogens, and thus contributing to the failure of all immunological functions and clinical collapse . While highly speculative, this scenario highlights the importance to understand m polarization in the context of immune activation and pathogen - driven disease, and its potential to be yet another convergence point targeted by mtb and hiv to circumvent the host immune system . 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.
Sweet potatoes (ipomoea batatas) are ranked as the seventh most commonly consumed carbohydrate - rich food source in the world and one of the most important food crop in developing countries after rice, wheat, maize, and cassava . It is a high yielding economic crop with over 90% of global production cultivated in developing countries . Compared to other crops, sweet potato is considered to be a superfood, with high nutritional value and may be a better choice for consumption compared to potatoes (solanum tuberosum). In the caribbean, jamaica is the leading producer of sweet potatoes . It is a major component of the jamaican diet, where over 95% of the annual production (25,797,000 kg) is consumed locally as a source of digestible carbohydrate . It is estimated that over 50% of the population (1.35 million) consume sweet potato at least once per week as part of their diet in a boiled, roasted, fried, or baked form . However, despite a dietary preference for sweet potatoes in jamaica and other caribbean countries, studies have indicated that complex carbohydrate - rich foods may have high glycemic indices resulting in potential harmful health effects [57] and the development of chronic diseases . Excess consumption of high glycemic index foods can lead to hyperinsulinemia, insulin resistance, weight gain, and possibly obesity, leading to insulin - resistant syndrome [810]. Recent studies have shown a positive correlation between the consumption of foods with high glycemic index and increased risk of chronic diseases such as type 2 diabetes, cardiovascular diseases, and cancer [11, 12]. Other studies have shown that not all complex carbohydrate - rich foods have high glycemic index [13, 14]. In contrast, foods with a low glycemic index can be beneficial in reducing the incidences of chronic diseases [13, 15]. Despite the name, sweet potato may be beneficial to persons with type 2 diabetes, resulting from the high fiber and manganese content, which could aid in stabilizing blood sugar levels and reduce insulin resistance . However, little information is available on the glycemic indices of sweet potatoes and their impact on blood glucose and glycemic response after consumption . As such, the present study was undertaken to investigate the effect of different processing methods on the gi and glycemic responses of ten sweet potato cultivars that are commonly eaten in jamaica . The study was carried out using standard glycemic index testing protocol as outlined by wolever et al . Glucose was used as the reference food with a gi score of 100, tested in the subjects at baseline, midway, and at the end of the study . Subjects were appraised both verbally and in writing of the study protocol, and all gave written informed consent before participation . Ethics approval was granted by university hospital of the west indies ethics committee and conducted in accordance with its rules and regulations . Recruitment took place between february 2008 and march 2008 during which subjects were screened for any illness at the university of the west indies health center . Anthropometric data and lifestyle factors were derived from questionnaires . Only nondiabetic individuals between the ages of 25 and 45 years were eligible to participate in the study . Emphasis was placed on subjects who were healthy, with an active lifestyle, without any diagnosed diseases, and not on prescribed medication . During the study, subjects were advised to continue their customary daily activities without any change in their physical activities . Freshly harvested, matured tubers from the ten most commonly eaten sweet potato cultivars (dor, quarter million, yellow belly, ganja, watson, clarendon, minda, ms mac, eustace, and fire on land) were collected from a local farm in st . The proximate compositions of the sweet potatoes were determined using the standard aoac methods and the available carbohydrate content calculated by difference [13, 19]. Samples used for the gi studies were thoroughly washed then cooked by boiling, roasting, baking, or frying on the day of glycemic index testing . Foods processed by frying were peeled and cut into 50 grams wet weight available carbohydrate portions . They were then cut to 10 mm thickness (sweet potato wedges) and submerged in preheated, cholesterol - free vegetable cooking oil (lider brand, manufactured in jamaica), until slightly brown . Foods processed by roasting were washed and cooked (skin intact) using preheated charcoal for 45 minutes in an open system . Foods processed by baking were washed and cooked (skin intact) in a preheated electric oven at 175c for 45 minutes . Foods processed by boiling were washed, peeled, and cut into 25 mm slices . They were then cooked in water (gentle boiling) with the lid of the cooking vessel on for 20 minutes, followed by simmering heat (lid of cooking vessel off) for a further 10 minutes . After the boiling process, the available carbohydrate content was determined, to assess the loss of sugars that may have occurred during cooking . The foods were then cut into 50 grams available carbohydrate portions, required for gi analysis . A randomized cross - over study design was conducted with 10 healthy nondiabetic subjects (5 males and 5 females). Fifty grams (50 g) available carbohydrate portions of the test foods were administered to the subjects on separate mornings after a 1012-hour overnight fast . For individual subjects subjects were asked not to perform any strenuous activities, take long walks, or consume alcohol on the day of glycemic index determination . Test meals were consumed within 10 minutes and supplemented with 250 ml of water . Capillary pricked - finger blood samples were taken (3 - 4 drops) at baseline (0 mins), 15, 30, 45, 60, 90, and 120 minutes after the meal was consumed . Blood glucose was determined using the glucose oxidase method using a uv / visible ultraspec spectrophotometer (model 1100 pro). The incremental areas under the curve (iauc), excluding the area beneath the fasting level, was calculated geometrically . The gi was then calculated by expressing the glycemic response area for the sweet potato as a percentage of the mean response area of the reference food (glucose) taken by the same subjects . The power of the tests with 10 subjects was expected to have 80% power to detect differences in glycemic response of about 20% in the incremental areas under the glucose response curves (iaucs) above the fasting level between the foods . Statistical analyses were performed using the statistical package for social sciences version 12.0 (spss inc ., the changes in blood glucose levels after consumption of the different sweet potato varieties by time interval were analyzed by repeated - measurement analysis of variance (anova) and duncan's multiple range tests . The 10 subjects (5 males and 5 females), all of african descent, were between ages 25 and 45 years with a mean age of 27 2 years and bmi ranging from 22.91 kg / m to 28.32 kg / m (24.65 0.4 kg / m). The carbohydrate content of the unprocessed sweet potato tubers ranged from 26.86 [g/100 g] to 31.74 [g/100 g] with fire on land having the lowest and minda the highest content . Dietary fiber content of the ten varieties also differed significantly ranging from 2.87 0.03 [g/100 g] to 3.74 0.04 [g/100 g] (table 1). Actual serving sizes containing 50 g available carbohydrate [g/100 g] for the test meals were larger for boiled foods than those roasted, baked, or fried (table 2). Table 3 shows the glycemic index values of the different sweet potato varieties calculated relative to the reference food (glucose gi = 100) and classified as high (70 to 100), intermediate (55 to 69), or low (<55). The gi values were significantly lower for foods processed by boiling (ganja variety having the lowest gi = 41 5) when compared to the other processing methods (p <0.05). Foods baked and roasted had high gi values, while those fried (sweet potato wedges) had intermediate to moderately high gi values (63 2 to 77 4). Table 4 shows the incremental area under the glucose response curve for the sweet potato varieties studied, while figure 1 shows the mean glycemic responses of all the sweet potato cultivars processed by the different cooking methods . The classification of foods based on their glycemic index has dispelled the repeatedly suggested dietary notion that carbohydrate - rich foods have deleterious health effects, and, as such, consumption should be limited [20, 21]. In fact, there are numerous evidence - based studies which dismiss the negative view of carbohydrate - rich foods and clearly demonstrate that not all carbohydrates are created equal furthermore, variations in the physiochemical properties of complex carbohydrates have been shown to elicit dissimilar physiological effects when consumed . Also, some complex carbohydrate - rich foods are undeniably beneficial and do not cause blood glucose levels to spike any greater than some simple sugars . However, food preparation is important and should be considered, as the method of cooking can alter the structure and nature of the starches resulting in significant effects on postprandial blood glucose responses . The results from this study show that the processing of sweet potatoes by boiling illicits lower gi values when compared to frying, baking, and roasting (table 3). This may be linked to the chemical structure of starches, that is, the amylose - amylopectin ratio . Reported that rice with higher amylose content was accompanied by lowered metabolic response and lower gi values . Boiling is believed to induce gelatinization, thereby permanently disrupting the amylose - amylopectin structure of the starch complex, thus making it more readily accessible by digestive enzymes . At the same time retrograded amylose is indigestible due to the presence of stronger hydrogen bonding in comparison with retrograded amylopectin . Concomitantly, greater amounts of resistant starches (rs1, rs2, and rs3) may have been retained in the boiled foods . Furthermore, as these foods cool, the possibility of forming r3-resistant starches (retrograded starches) increases . This occurs as the starches undergo recrystallization due to the formation of intermolecular hydrogen bonds . Other resistant starches (r1 and r2) present in the foods after the leaching of free sugars during the boiling process also play a role in retarding the enzymatic degradation of the starches, thus reducing the glycemic response . In a similar study, englyst and cummings reported that about 7% of starch in reheated boiled potatoes (solanum tuberosum sp .) Escapes digestion in the ileum compared with about 3% in freshly cooked potato . Termed sweet potato wedges and is a popular alternative to french fries (irish potatoes solanum tuberosum). The results indicate that fried sweet potatoes had intermediate to moderately high gi (table 4). The gi of ms mac variety was similar to that of french fries (solanum tuberosum sp .) The lower glycemic indices observed on frying compared to baking and roasting could be attributed to the increased fat content resulting in retardation in starch degradation, consequently delaying gastric emptying and glycemic response . This principle was supported by fernandes et al . Who reported similar gi values for french fries from solanum sp . In addition, in vitro studies revealed that the frying process increases the amount of rs in potatoes . This decreases the rate of hydrolysis of the amylose - amylopectin starch structure resulting in a lowered glycemic response . In addition, studies by holm et al . And leeman et al . Suggested that amylose is prone to react with lipids to form amylose - lipid complexes thus reducing the rate of amylolysis and resulting in lower glycemic responses and gi values . The study also shows that cooking by roasting and baking resulted in spikes in postprandial blood glucose levels for all the sweet potato varieties studied (8194) as seen in the iauc in table 4 . Cooking with the skin intact increases the availability of free sugars which are immediately hydrolyzed by salivary amylase and are instantly absorbed . Additionally the absence of a water - rich environment as similar to that of boiling would have resulted in less starch gelatinization and by extension production of lower levels of retrograded / rs 3 starch . Additionally, the gi values of these sweet potato cultivars studied could be substantiated by correlating the texture of the sweet potato cultivars as described by studies done by henry et al . Who reported a strong positive correlation between gi and the texture rating in commercially available potatoes eaten in great britain . Furthermore, intravarietal variations could be related to differences in the starch physicochemical properties and maturity index of the different varieties used . Additionally, it has been reported that precooking or allowing the food to cool and then reheating before consumption may elicit a lower glycemic response compared with consumption immediately after cooking [28, 33]. As such, further studies can be carried out to investigate the effects of starch properties, maturity index, precooking, cooling, and reheating on these sweet potatoe cultivars on gi . Since sweet potato is a major staple in the jamaican and the wider caribbean diet, the results suggest that health conscious individuals and persons with diabetes should consider avoiding the consumption of baked or roasted sweet potatoes and should not be misguided by the confounding fact that it is a highly nutritious root vegetable . Similarly, the identification of cooking methods of sweet potatoes with lower glycemic responses may help reduce the gi and glycemic load of the jamaican diet which could prove beneficial in the management and prevention of other chronic diseases . This study is the first to report variations in the glycemic indices and blood glucose responses among the sweet potato (ipomoea batatas) cultivars eaten in jamaica and the wider caribbean with respect to different cooking methods . Tubers processed by boiling had the lowest glycemic, index while those roasted and baked had significantly higher glycemic indices . Generally ganja had the overall lowest glycemic index and eustace the highest among the sweet potato varieties studied . The results therefore indicate that method of food preparation significantly impacts on the glycemic index of jamaican sweet potatoes (p <0.005). Consumption of boiled sweet potatoes may minimize the risk of postprandial blood glucose spikes, thereby reducing diabetic and cardiovascular disease indices and thus may prove to be more efficacious in the management of type 2 diabetes mellitus . Increased amount of hydrogenated fats in the diet is potentially unhealthy; hence, it is advisable to limit the consumption of fried foods.
Malaria, the parasitic disease of the red blood cell caused by plasmodium sp, kills more than one million children each year in africa alone (kwiatkowski, 2005). The strong selective pressure that malaria exerts has been shaping the human genome for at least 6,000 years (rich et al, 1998) and the disease is the driving force behind several genetic disorders, such as the sickle cell trait, glucose-6-phosphate dehydrogenase deficiency and thalassemia (kwiatkowski, 2005). The single nucleotide polymorphisms (snps) responsible for the above traits afford protection against malarial parasites and hence have been maintained in some human populations by a mechanism of balancing selection (boldt et al, 2006). In addition to the erythrocyte variants, snps in genes that modulate the immune response have been also reported to confer resistance to malaria (kwiatkowski, 2005). Different human populations may have developed independent and different responses to malaria, but the bulk of studies on the genetics of those responses has been performed in africa, where remarkable examples of differences in protection can be found (kwiatkowski, 2005). Most of the sub - saharan african populations, for instance, are completely resistant to the malaria caused by p. vivax, whereas populations outside africa are not . The protection in africans is believed to be associated with a snp in the promoter region of the fy gene (tournamille et al, 1995), which abolishes transcription in erythrocytes and consequently prevents the parasite from invading the red blood cells (miller et al, 1976). Malaria is also endemic in south asian countries such as india, nepal, bhutan, and sri lanka, (kondrashin, 1992). In nepal, for example, the who reported 4,637 cases in 2004, from a population of 27.3 million; 7.9% of the cases were caused by p. falciparum (http://w3.whosea.org/en/section10/section21/section340.htm). However, the probable number of cases was judged to be an order of magnitude higher than the reported number, leading to an estimated incidence of approximately 0.17% per year . Few studies of resistance have been performed in this region (wattavidanage et al, 1999). The lack of data on malaria - protective genes in south asia is surprising since some ethnic groups, such as the tharu in nepal, have long been known to be resistant to the disease (terrenato et al, 1988). A large part of nepal consists of mountains where malaria cannot be transmitted (gillies, 1988), but the southern part of the country, the terai area where malaria cases are concentrated is flat and exposed to the malaria parasites p. vivax and p. falciparum (sherchand et al, 1995). The incidence of malaria in the terai has restricted the settlement of the region, but nevertheless it has been inhabited by aboriginal groups since pre - historical times (van driem, 2001). Besides these lowland inhabitants, there are other nepalese populations who live up in the hills but visit low lying areas in the terai during the daytime when the mosquitoes that spread malaria do not bite a pattern referred to as diurnal uphill - downhill migration (van driem, 2001). The terai has also experienced migration from other parts of the country during the rana period, which started in 1856 and led to deforestation of the region, and from the 1950s with the implementation of a malaria eradication program (terrenato et al, 1988). It has been proposed that the high frequency of thalassemia among some nepalese populations such as the tharu and the danuwar could explain their biological resistance to malaria (modiano et al, 1991; sakai et al, 2000). However, the involvement of other genes in this protection and the resistance in other populations have not been thoroughly investigated (matsuoka et al, 2003). We have therefore examined the frequency of seven malaria - protective snps in 928 healthy individuals from different parts of nepal in order to investigate whether known african protective alleles also confer resistance in asia . By comparing populations living successfully in malaria - endemic lowland regions, and thus resistant to the disease, with those from malaria - free highland regions, we could investigate the contribution these alleles make to malaria resistance in this region . The sample consisted of 928 healthy individuals representing different groups identified on the basis of ethnicity, language and geography within nepal (van driem, 2001; kraayenbrink et al, 2006). All samples were collected in accordance with the human genome diversity project ethics protocol and with the appropriate approval and cooperation of local institutions . The research aims of the project were explained in nepali to each individual and informed consent forms in both nepali and english were signed (van driem, 2001; kraayenbrink et al, 2006). Rural, rather than urban, areas were sampled, both parents belonged to the same ethno - linguistic group as the donor, and the donor's name was characteristic of the ethnic group and the geographic location within nepal . Seven snps located in the genes hbb, fy, g6pd, tnfsf5, tnf, nos2, and fcgr2a were selected based on previous reports of association with malaria resistance in non - south asian patients (kwiatkowski, 2005) and amplified by polymerase chain reaction (pcr). Primers were designed with primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), screened for primer - dimer and hairpin interactions using autodimer (vallone and butler, 2004) and combined into two multiplex reactions, m1 (fy, tnfsf5, g6pd and hbb) and m2 (tnf, nos2 and fcgr2a) (table 1). Amplification was carried out in a 25 l volume containing 10 ng genomic dna, 150 m of deoxynucleotide triphosphates (dntps; amersham biosciences), 1 mm mgcl2, 2.0 u of platinum taq dna polymerase (invitrogen, paisley, uk) with 1 x buffer, and the primer final concentrations shown in table 1 . The thermal conditions in an mj research dna engine tetrad (genetic research instrumentation, braintree, uk) were as follows: denaturation at 94c for 15 min, followed by 15 cycles of touchdown pcr: 94c for 30 sec, 65c for 30 sec, 72c for 45 sec, with a 1c decrease in annealing temperature in every cycle, and then 17 cycles of standard pcr (94c for 30 sec, 50c for 30 sec, 72c for 45 sec), followed by extension at 72c for 7 min and storage at 4c . Unincorporated dntps and primers were removed by mixing 15 l of these pcr products with a 5 l volume containing 2.0 u of exonuclease i (usb, cleveland, usa) and 5.0 u of shrimp alkaline phosphatase (or sap; usb) for 1 hr at 37c followed by incubation at 80c for 15 min . Pcr primer sequences for the multiplex amplification of 7 snps previously associated with resistance to malaria the protective allelic state is listed first . Both forward and reverse pcr primers had been previously published (tishkoff et al, 2001) the snps in bold were polymorphic in the studied population . Seven mini - sequencing primers were selected from either the reverse or forward direction for each locus and screened for hairpin and primer - dimer interactions as described above . The snps at fy, tnfsf5, g6pd and hbb (multiplex 1) and those at tnf, nos2 and fcgr2a (multiplex 2) were genotyped in two separate mini - sequencing reactions using the abi prism snapshot kit (applied biosystems, foster city, usa). The reactions were carried out in a total volume of 5 l containing 2.5 l of snapshot multiplex ready reaction mix, 1.0 l of the minisequencing primer mix 1 or 2 (table 2), 1.0 l of deionized water and 0.5 l of the purified pcr products . Thermal cycling conditions consisted of 25 cycles of 96c for 10 sec, 50c for 5 sec, and 60c for 30 sec . Unincorporated labeled dideoxynucleotide triphosphates (ddntps) were removed by adding 1u of sap to the minisequencing products, and incubated at 37c for 1 hr, followed by 15 min at 80c . One tenth of the purified minisequencing reaction (0.5 l) was mixed with 9.0 l hi di formamide and 0.5 l genescan-120 liz size standard (applied biosystems, warrington, uk). This mixture was then denatured for 2 min at 96c and analysed in the abi prism 3100 genetic analyzer, using filter set e5 and a 36 cm capillary array filled with pop4 polymer (applied biosystems). Minisequencing primers for the multiplex detection of 7 snps previously associated with resistance to malaria note: the mini - sequencing primers for the underlined genes were chosen based on the reverse strand and the scored alleles should be the reverse of those shown in table 1 . Allele and genotype frequencies were obtained by direct counting and differences in allele frequencies were assessed by means of fisher's exact test in the 928 healthy individuals residing in either lowlands (n = 146) or highlands (n = 782) of nepal . The hypothesis of hardy - weinberg equilibrium (hwe) was tested using the approach of guo and thompson implemented in arlequin 2.0 (schneider et al, 2000). Conventional pairwise fst distances and analysis of molecular variance (amova) were obtained with arlequin for the 18 populations whose sample size was greater than 20 . For amova, populations were pooled according to whether they reside in lowlands or highlands, and the results were then compared with no grouping . Differences in allele frequencies for the 18 populations were assessed by the mann - whitney test using spss v 14.0 . The sample consisted of 928 healthy individuals representing different groups identified on the basis of ethnicity, language and geography within nepal (van driem, 2001; kraayenbrink et al, 2006). All samples were collected in accordance with the human genome diversity project ethics protocol and with the appropriate approval and cooperation of local institutions . The research aims of the project were explained in nepali to each individual and informed consent forms in both nepali and english were signed (van driem, 2001; kraayenbrink et al, 2006). Rural, rather than urban, areas were sampled, both parents belonged to the same ethno - linguistic group as the donor, and the donor's name was characteristic of the ethnic group and the geographic location within nepal . Seven snps located in the genes hbb, fy, g6pd, tnfsf5, tnf, nos2, and fcgr2a were selected based on previous reports of association with malaria resistance in non - south asian patients (kwiatkowski, 2005) and amplified by polymerase chain reaction (pcr). Primers were designed with primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), screened for primer - dimer and hairpin interactions using autodimer (vallone and butler, 2004) and combined into two multiplex reactions, m1 (fy, tnfsf5, g6pd and hbb) and m2 (tnf, nos2 and fcgr2a) (table 1). Amplification was carried out in a 25 l volume containing 10 ng genomic dna, 150 m of deoxynucleotide triphosphates (dntps; amersham biosciences), 1 mm mgcl2, 2.0 u of platinum taq dna polymerase (invitrogen, paisley, uk) with 1 x buffer, and the primer final concentrations shown in table 1 . The thermal conditions in an mj research dna engine tetrad (genetic research instrumentation, braintree, uk) were as follows: denaturation at 94c for 15 min, followed by 15 cycles of touchdown pcr: 94c for 30 sec, 65c for 30 sec, 72c for 45 sec, with a 1c decrease in annealing temperature in every cycle, and then 17 cycles of standard pcr (94c for 30 sec, 50c for 30 sec, 72c for 45 sec), followed by extension at 72c for 7 min and storage at 4c . Unincorporated dntps and primers were removed by mixing 15 l of these pcr products with a 5 l volume containing 2.0 u of exonuclease i (usb, cleveland, usa) and 5.0 u of shrimp alkaline phosphatase (or sap; usb) for 1 hr at 37c followed by incubation at 80c for 15 min . Pcr primer sequences for the multiplex amplification of 7 snps previously associated with resistance to malaria the protective allelic state is listed first . Both forward and reverse pcr primers had been previously published (tishkoff et al, 2001) the snps in bold were polymorphic in the studied population . Seven mini - sequencing primers were selected from either the reverse or forward direction for each locus and screened for hairpin and primer - dimer interactions as described above . The snps at fy, tnfsf5, g6pd and hbb (multiplex 1) and those at tnf, nos2 and fcgr2a (multiplex 2) were genotyped in two separate mini - sequencing reactions using the abi prism snapshot kit (applied biosystems, foster city, usa). The reactions were carried out in a total volume of 5 l containing 2.5 l of snapshot multiplex ready reaction mix, 1.0 l of the minisequencing primer mix 1 or 2 (table 2), 1.0 l of deionized water and 0.5 l of the purified pcr products . Thermal cycling conditions consisted of 25 cycles of 96c for 10 sec, 50c for 5 sec, and 60c for 30 sec . Unincorporated labeled dideoxynucleotide triphosphates (ddntps) were removed by adding 1u of sap to the minisequencing products, and incubated at 37c for 1 hr, followed by 15 min at 80c . One tenth of the purified minisequencing reaction (0.5 l) was mixed with 9.0 l hi di formamide and 0.5 l genescan-120 liz size standard (applied biosystems, warrington, uk). This mixture was then denatured for 2 min at 96c and analysed in the abi prism 3100 genetic analyzer, using filter set e5 and a 36 cm capillary array filled with pop4 polymer (applied biosystems). Minisequencing primers for the multiplex detection of 7 snps previously associated with resistance to malaria note: the mini - sequencing primers for the underlined genes were chosen based on the reverse strand and the scored alleles should be the reverse of those shown in table 1 . Allele and genotype frequencies were obtained by direct counting and differences in allele frequencies were assessed by means of fisher's exact test in the 928 healthy individuals residing in either lowlands (n = 146) or highlands (n = 782) of nepal . The hypothesis of hardy - weinberg equilibrium (hwe) was tested using the approach of guo and thompson implemented in arlequin 2.0 (schneider et al, 2000). Conventional pairwise fst distances and analysis of molecular variance (amova) were obtained with arlequin for the 18 populations whose sample size was greater than 20 . For amova, populations were pooled according to whether they reside in lowlands or highlands, and the results were then compared with no grouping . Differences in allele frequencies for the 18 populations were assessed by the mann - whitney test using spss v 14.0 . We typed seven snps in genes previously associated with protection to malaria in africa (kwiatkowski, 2005), namely hbb, fy, g6pd, tnfsf5, tnf, nos2, and fcgr2a, in 928 healthy individuals from nepal, a country in asia where the disease is also endemic . Out of the seven snps, five were found to be fixed for the non - protective allele at hbb, fy, g6pd, tnfsf5 and nos2 (table 1). Surprisingly, the protective allele at rs5030868 in g6pd which has been observed in other south asian countries, such as india (balgir, 2006) was absent from the large nepalese sample examined here . The non - protective allele at these snps is also fixed in all hapmap samples, with the exception of africans (http://www.hapmap.org/index.html.en), and also in non - africans from dbsnp (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp). On the other hand, the snps in tnf (rs1800629) and fcgr2a (rs1801274) were polymorphic in the nepalese population, and the protective alleles were found at an overall frequency of 93% and 27%, respectively . In the combined nepalese sample, the genotype frequencies at these snps were found to depart significantly from hwe (p<0.0001). We then investigated the correlation of these two snps with the regions in nepal that are exposed to malaria or not, i.e. Lowlands and highlands . The frequency of the protective allele at tnf in our entire data set (n=928) was 89% in the lowlands and 94% in the highlands, and the difference in this allele frequency was statistically significant (fisher's exact test, p=0.02). In combined populations from the highlands a significant departure from hwe was observed (p=0.0002), whereas in the lowlands the hypothesis of random association of alleles was not rejected (p=0.0679). For fcgriia, no significant difference was seen in the frequency of the protective allele between lowland (22%) and highland (28%) populations; the genotype frequencies in the combined populations from the highlands departed from hwe (p=0.001) but those in the lowlands did not (p=0.326). Subsequently we looked at the correlation of these snps with malaria exposure in the 18 individual populations of nepal which had a sample size greater than 20 (n=586, figure 1). No significant departures from hw expectations were found in individual populations (table 2), which suggested that the p values obtained above reflected population subdivision present in the country . Red lines indicate populations from malaria - endemic lowland regions, black lines from malaria - free highland regions . Then, it was surprising to see among the individual populations that for tnf (rs1800629), four out of five populations living in malarial areas actually had lower frequencies of the protective allele than populations living in malaria - free regions (figure 1a). Two groups showed frequencies greater than 25% of the non - protective allele at rs1800629, the sherpa and the danuwar (figure 1a, table 3). Both of them live in the malaria - free highlands of nepal, which could explain the unconstrained higher frequency of the non - protective allele . However, it is striking that some of the other highland populations are fixed or almost fixed for the protective allele (figure 1a, table 3). For fcgriia (rs1801274), a similar lack of association of the protective allele with malarial regions of nepal was observed (fig . A frequency greater than 52% of the protective allele was seen in two populations living at high altitude, high caste newar and chantyal, whereas this allele was found to be as rare as 18% in kumal and majhi bote, two groups exposed to the disease (figure 1b, table 3). However, the differences in allele frequencies between the highland and lowland populations were not statistically significant at either tnf or fcgriia (mann - whitney u test). This lack of correlation between the protective alleles at both tnf and fcgriia and the distribution of malaria suggests that malaria did not select for these alleles in nepal . Genotype and allelea frequencies in 18 populations from lowlands and highlands of nepal frequency of the protective alleles g and c at tnf and fcgriia, respectively p values after testing for departure from hardy - weinberg equilibrium (random association of alleles in diploid individuals) the populations in bold live in the malarial lowlands of nepal . The remaining groups live in regions of high altitudes, which are malaria - free finally, we assessed whether or not the inter - population diversity between groups living in malaria - free regions and those exposed to the disease varied significantly . By means of amova, we found that 91% and 95% of the entire variation at rs1800629 (tnf) and rs1801274 (fcgriia), respectively, was observed within populations . In contrast, the amount of variation between groups was zero for tnf and 0.54% (p=0.22; not significantly different from zero) for fcgriia, whereas the fst (distance between populations among groups) was 9% for tnf and 5% for fcgriia . The observed low fst values and the amova analyses further support our conclusion that it is unlikely that selection by malaria has significantly influenced the frequencies of the protective alleles at tnf and fcgriia in nepal . Although genes such as g6pd, tnf and fcgriia have previously been associated with malaria resistance in parts of asia (balgir, 2006; matsuoka et al, 2003; omi et al, 2002; wattavidanage et al, 1999), the disease did not play an important role in selecting or maintaining the protective alleles of these genes in the populations that we have examined in nepal . Recent migration and admixture could equalise gene frequencies between populations, but both our sampling strategy and the high differentiation between populations observed at other loci (results not shown) lead us to conclude that such factors cannot explain our results . It is possible that selection for other functions of these genes has dominated in nepal, but perhaps more likely either that the populations and disease have not co - existed in this region for long enough for malaria to influence allele frequencies detectably, or that genetic drift dominates to such an extent in these small populations (parkin et al, in press) that it overwhelms any selective force by malaria . The investigation of additional snps and genes that may contribute to malaria resistance, especially those involved in the human immune response, could help to distinguish between the above hypotheses.
Pyogenic spinal infections encompass the spectrum of but are not limited to spondylitis, discitis, spondylodiscitis, and epidural abscess . The incidence of pyogenic spine infections is increasing due to a greater number of patients with immunosuppression and bacteremia . In our experience, pyogenic spine infections are also prevalent in the end - stage renal disease population . While an epidural abscess with a neurological deficit is a clear indication for surgery, there is no consensus on the type of intervention that best manages spondylodiscitis . There have been some attempts to suggest an algorithm or guidelines for the management of these cases . Treatment either involves a computed tomography (ct)-guided biopsy with antibiotics or extensive surgery with spinal reconstruction and instrumentation . Traditionally, a laminectomy was ineffective because removal of the posterior elements destabilized the spine in the setting of disc destruction, and access to the actual disk was limited . Minimally invasive spine surgery (mis) allows for reduction in blood loss, length of stay, recovery time, and complications . The benefits of mis are all highly desirable features when treating patients with a suspected infection, especially since these patients are often sick or immunocompromised with multiple comorbidities . This paper reports a series of patients with spinal infection in whom mis techniques were employed to obtain a quick diagnosis to enable appropriate antibiotic therapy and faster recovery from pain, reduce the disease burden, and avoid a potentially bigger operation . A retrospective chart review of all patients who underwent mis surgery for discitis, spondylodiscitis, or epidural abscess by the senior surgeon (hd) was collected from january 2014 to september 2015 . Preoperative data collected included prior history of infection, preoperative visual analog score (vas) scores, symptoms at the time of presentation, and any neurologic deficit . The surgical procedure performed was detailed including estimated blood loss (ebl), operative time, and the bacterial organism cultured . The patients were placed prone, and fluoroscopy was used to localize the correct level . A lateral to medial angulation of the tubular retractor allowed for exposure of the disk space without retraction of the dura . The midline structures were preserved, and a lateral laminectomy and partial facetectomy were done using a high - speed drill . Culture swabs were used to obtain cultures, and pathology specimen was sent . Up biting pituitary rongeurs and curettes were used to debride necrotic disc material, and the disk was irrigated with antibiotic irrigation . The wound was closed in a standard fashion with vicryl sutures and dermabond for the skin [figures 13]. Sagittal magnetic resonance imaging showing t7t8 spondylodiscitis with an epidural abscess (a) operative room setup for minimally invasive surgery and (b) transpedicular discectomy using tubular retractors and removal of pus () from the disc space postoperative computed tomography scan showing the minimally invasive approach and trajectory of surgery (arrows) with excellent outcome the patients were placed prone, and fluoroscopy was used to localize the correct level . A lateral to medial angulation of the tubular retractor allowed for exposure of the disk space without retraction of the dura . The midline structures were preserved, and a lateral laminectomy and partial facetectomy were done using a high - speed drill . Culture swabs were used to obtain cultures, and pathology specimen was sent . Up biting pituitary rongeurs and curettes were used to debride necrotic disc material, and the disk was irrigated with antibiotic irrigation . The wound was closed in a standard fashion with vicryl sutures and dermabond for the skin [figures 13]. Sagittal magnetic resonance imaging showing t7t8 spondylodiscitis with an epidural abscess (a) operative room setup for minimally invasive surgery and (b) transpedicular discectomy using tubular retractors and removal of pus () from the disc space postoperative computed tomography scan showing the minimally invasive approach and trajectory of surgery (arrows) with excellent outcome a total of 7 patients were identified as having undergone mis procedures for the treatment of spinal infection . The mean age was 60.1 years (median 60 years, range 5569) and there were five males and two females . Four patients had symptoms of only back pain, two had back pain and bilateral leg pain, and one had back pain with left leg pain . Six of the 7 patients were neurologically intact and one patient had a foot drop . Three patients had known bacteremia at the time of surgery, two patients had pneumonia, and one patient had a urinary tract infection and had lower extremity cellulitis . Of the seven patients, three had positive cultures (blood / urine) before surgical intervention . One patient had a prior interventional radiology - guided biopsy of the infectious disc space at an outside institution, but no positive cultures were identified . Five patients had infections of the lumbar spine and 2 patients in the thoracic spine . The average number of levels operated on in every surgery was 1.1 0.4 levels . Surgical details including the operation preformed, ebl, and operative time are shown in table 1 . The average ebl was 38 35 ml, average operative time was 77.6 39.2 min . In 6 of the 7 cases, we were able to obtain positive cultures . Details of preoperative sedimentation rate, c - reactive protein, white blood cell count, organism identified, antibiotic, and treatment duration are shown in table 2 . Patient demographics and surgical procedure, estimated blood loss (ebl), and operative time patient preoperative and postoperative laboratory findings and antibiotic regimen five patients had follow - up 29 months after surgery . The diagnosis of a spinal infection is made based on clinicoradiological evidence and obtaining appropriate cultures . Up to half of these infections can be caused by methicillin - resistant s. aureus . This is followed by aerobic and anaerobic streptococcus, escherichia coli, pseudomona aeruginosa, streptococcus pneumonia, and enterobacter . Ct - guided cultures are often negative, especially when patients have been on empirical preprocedural antibiotics for a suspected infection . A negative culture result may call into doubt the presence of infection and may require treatment with broad spectrum antibiotics not optimized to the actual pathogen . Yang et al . Showed that 90% of their patients obtained cultures when this was done with a percutaneous endoscopic technique as opposed to the causative bacteria being identified in <50% of their cases where a ct - guided biopsy was performed . Heyer et al . Showed that ct - guided biopsy's bacteriological yield was only 32% . We were able to obtain a positive culture in all but one of our patients, despite almost all of them being on antibiotics before surgery . Adequate tissue for histopathology and cultures not only determines the correct bacteria but also helps rule out tuberculosis, sterile discitis, and fungal or parasitic spinal infections . One of our patients (not included) suspected of having pyogenic discitis was eventually diagnosed to have gout of the disk based on pathology specimen, which not only provided the correct diagnosis and pain relief after starting her on appropriate medication but also avoided long - term antibiotics . Some studies report up to 98% of patients presented with local spine pain of longer than 6 weeks' duration, and 50% with fever at presentation . Single - level noncomplicated spondylodiscitis, surgical stabilization with percutaneous screw, and rod stabilization was associated with faster recovery, lower pain scores, and improved quality of life compared with thoracolumbar bracing . Chen et al . Reported quick pain relief from a vas of 9.22.3 after mis endoscopic surgery and antibiotic treatment for spondylodiscitis in a cohort of immunocompromised patients . Similarly, another group too reported that all patients showed immediate back pain reduction after surgery for discitis . Apart from allowing for bacteriological and histological testing, mis techniques enable drainage of infected material, prompt relief of pain and suffering, and early patient mobilization . Our cases illustrate that even in the presence of discitis without an epidural abscess, it is possible to use mis techniques to drain infected material and result in immediate improved functional recovery . Mis techniques allow for a less invasive approach that may be appropriate for patients with extensive other comorbidities that exclude a larger surgical approach . Mis techniques further reduce blood loss, pain resulting in early postoperative mobilization, and shorter hospital stay and recovery time . Other minimally invasive options such as percutaneous endoscopic lavage and drainage have been reported to be successful in obtaining a bacteriologic diagnosis, relieving the patient's symptoms, and assisting in the eradication of spondylitis . While percutaneous endoscopic techniques are not widely used by all surgeons, we do believe that mis techniques and surgery through a tubular retractor is a familiar technique to many spine surgeons . The same mis techniques can be applied to drain and obtain tissue for lumbar and thoracic discitis patients . Some authors have described an alternative technique for the surgical treatment of lumbar discitis and osteomyelitis using a direct lateral retroperitoneal approach, which allows for thorough debridement and anterior column reconstruction while avoiding the need to mobilize the great vessels . State that surgeons who are comfortable with the direct lateral retroperitoneal approach for degenerative pathology should exercise caution when adapting this approach to infectious cases . Local anatomy is often distorted by the infection, and the disc space may not be readily identifiable because it is necrotic and inadequate . The distorted anatomy makes it easier to stray from the disc space and encounter bleeding from a segmental vessel or the great vessels . Nonetheless, the technique allows for effective eradication of infection, with reasonable blood loss and minimal approach - related morbidity . In cases of discitis, which are often managed with a biopsy and antibiotics, early debridement of these infections by percutaneous discectomy can accelerate the natural process of healing and prevent progression to bone destruction and epidural abscess and delayed treatment may result in serious neurologic complications . This series demonstrates that mis techniques do not result in an increased rate of secondary surgery and in fact arresting the infectious process early may prevent further bone destruction and the need for subsequent surgery . The diagnosis of a spinal infection is made based on clinicoradiological evidence and obtaining appropriate cultures . Up to half of these infections can be caused by methicillin - resistant s. aureus . This is followed by aerobic and anaerobic streptococcus, escherichia coli, pseudomona aeruginosa, streptococcus pneumonia, and enterobacter . Ct - guided cultures are often negative, especially when patients have been on empirical preprocedural antibiotics for a suspected infection . A negative culture result may call into doubt the presence of infection and may require treatment with broad spectrum antibiotics not optimized to the actual pathogen . Yang et al . Showed that 90% of their patients obtained cultures when this was done with a percutaneous endoscopic technique as opposed to the causative bacteria being identified in <50% of their cases where a ct - guided biopsy was performed . Heyer et al . Showed that ct - guided biopsy's bacteriological yield was only 32% . We were able to obtain a positive culture in all but one of our patients, despite almost all of them being on antibiotics before surgery . Adequate tissue for histopathology and cultures not only determines the correct bacteria but also helps rule out tuberculosis, sterile discitis, and fungal or parasitic spinal infections . One of our patients (not included) suspected of having pyogenic discitis was eventually diagnosed to have gout of the disk based on pathology specimen, which not only provided the correct diagnosis and pain relief after starting her on appropriate medication but also avoided long - term antibiotics . Some studies report up to 98% of patients presented with local spine pain of longer than 6 weeks' duration, and 50% with fever at presentation . Single - level noncomplicated spondylodiscitis, surgical stabilization with percutaneous screw, and rod stabilization was associated with faster recovery, lower pain scores, and improved quality of life compared with thoracolumbar bracing . Chen et al . Reported quick pain relief from a vas of 9.22.3 after mis endoscopic surgery and antibiotic treatment for spondylodiscitis in a cohort of immunocompromised patients . Similarly, another group too reported that all patients showed immediate back pain reduction after surgery for discitis . Apart from allowing for bacteriological and histological testing, mis techniques enable drainage of infected material, prompt relief of pain and suffering, and early patient mobilization our cases illustrate that even in the presence of discitis without an epidural abscess, it is possible to use mis techniques to drain infected material and result in immediate improved functional recovery . Mis techniques allow for a less invasive approach that may be appropriate for patients with extensive other comorbidities that exclude a larger surgical approach . Mis techniques further reduce blood loss, pain resulting in early postoperative mobilization, and shorter hospital stay and recovery time . Other minimally invasive options such as percutaneous endoscopic lavage and drainage have been reported to be successful in obtaining a bacteriologic diagnosis, relieving the patient's symptoms, and assisting in the eradication of spondylitis . While percutaneous endoscopic techniques are not widely used by all surgeons, we do believe that mis techniques and surgery through a tubular retractor is a familiar technique to many spine surgeons . The same mis techniques can be applied to drain and obtain tissue for lumbar and thoracic discitis patients . Some authors have described an alternative technique for the surgical treatment of lumbar discitis and osteomyelitis using a direct lateral retroperitoneal approach, which allows for thorough debridement and anterior column reconstruction while avoiding the need to mobilize the great vessels . State that surgeons who are comfortable with the direct lateral retroperitoneal approach for degenerative pathology should exercise caution when adapting this approach to infectious cases . Local anatomy is often distorted by the infection, and the disc space may not be readily identifiable because it is necrotic and inadequate . The distorted anatomy makes it easier to stray from the disc space and encounter bleeding from a segmental vessel or the great vessels . Nonetheless, the technique allows for effective eradication of infection, with reasonable blood loss and minimal approach - related morbidity . In cases of discitis, which are often managed with a biopsy and antibiotics, early debridement of these infections by percutaneous discectomy can accelerate the natural process of healing and prevent progression to bone destruction and epidural abscess and delayed treatment may result in serious neurologic complications . This series demonstrates that mis techniques do not result in an increased rate of secondary surgery and in fact arresting the infectious process early may prevent further bone destruction and the need for subsequent surgery . This series shows that mis surgery techniques are safe and efficacious, reduce pain dramatically, and provide a high yield culture to guide appropriate antibiotic therapy in patients with thoracic and lumbar spondylodiscitis.
In june and september 2006, persons living in 56 villages of the 14 districts of tokat and sivas provinces (figure 1) who had a risk for cchfv infection other than occupational risk (i.e., healthcare, slaughterhouse work, and veterinary care) were randomly selected for the study . Villages and districts were selected based on residences of patients who were diagnosed with cchfv infection and treated at cumhuriyet university hospital, sivas, turkey, during the 2005 cchfv outbreak . Men and women were included in the study, but children <7 years of age were excluded because of difficulties in drawing blood samples and obtaining parental consent . Using epi info version 6 software (centers for disease control and prevention, atlanta, ga, usa) and assuming a cchfv seroprevalence of 10% in the study population with 99% confidence levels, we calculated error limits of 3% and a design effect of 1 . The estimated sample size required was 664, but the target sample size of high - risk persons was increased to 782 . Another 100 persons who were not at high risk for cchfv infection, but who lived in urban areas in the high - risk region and agreed to provide blood samples, were also included in the study . The cchfv seroprevalence study team in turkey included a physician and a nurse who went to the selected villages and approached the heads of the village and selected families . They explained the objectives of the study and asked for written informed consent from participants or parents of participating minors and then administered an interview - based questionnaire and collected a blood sample . The questionnaire considered the following variables: age; sex; history of tick bite, tick removal from animals, animal abortion, and animal slaughtering activity; close contact with a cchfv patient or an animal; and occupation . Blood samples (10 ml each) were collected and later tested for antibodies to cchfv by using immunoglobulin g (igg) elisa kits (vector - best; kolsovo, novosibirsk, russia). Spss version 10.0 (spss, chicago, il, usa) for windows software was used for statistical analysis . Univariate analysis was used to identify the risk factors for seropositivity of cchfv in the 782 participants . Of the 782 high - risk persons, forty - seven (12.1%) of 390 female participants and 53 (13.5%) of 392 male participants were seropositive for cchfv (p>0.05). Of the 100 serum samples collected in the urban population, only 2 (males 44 and 56 years of age) were seropositive . The cchfv seroprevalence in the 782 persons at high risk increased significantly with age (p<0.001). The highest proportion (23.5%) of seropositivity was found in persons 6170 years of age (p<0.001) (table 1). Figure 2 shows distribution of the cchfv seroprevalence in high - risk persons by age groups . The only variables significantly associated with presence of antibody against cchfv were history of tick bite (p = 0.002) or of tick removal from the animals (p = 0.03), employment in animal husbandry (p = 0.01) or farming (p = 0.02), and age> 40 years (p<0.001) (table 2). P value = 0.59 for persons living in rural area; for persons living in urban area, data are insufficient for statistical analysis . P value <0.001 for persons living in rural area; for persons living in urban area, data are insufficient for statistical analysis . Distribution of seroprevalence of immunoglobulin g against crimean - congo hemorrhagic fever virus by age groups for 782 high - risk persons living in rural areas of tokat and sivas provinces, turkey, 2006 . * cchfv, crimean - congo hemorrhagic fever virus . Serologic evidence of cchfv in turkey was reported in the 1970s (4). In 2003, the cchfv seroprevalence among 40 veterinarians in the tokat region was 2.5% (5). Another seroprevalence study conducted in 2003 among healthcare workers providing care to cchfv patients in turkey detected no seropositive persons (6). The present survey indicates that the seroprevalence of cchfv is higher in persons living in rural areas than in urban areas of the cchfv epicenter in turkey (12.8% vs 2.0%). However, because special markets for animal trading are located on the outskirts of large cities in iran, cchfv seroprevalence was found to be higher among persons living in urban areas than in persons living in rural areas of this country (7). Living in a rural area is a risk factor for exposure to the tick vector and for acquiring cchfv infection (8,9). Expected seroprevalence of cchfv among high - risk persons during epidemics has been found to be 10% (3); however, seroprevalence has been reported to be as low as 0.5% in nonepidemic situations (10). Other studies conducted in rural parts of iran and senegal during epidemics showed that the cchfv seroprevalence was 13%, comparable to our findings (9,11). In the present study, history of tick bite and history of tick removal from animals the overall tick - bite frequency was 62% (483/782) among persons at high risk and has been reported among 40%60% of cchfv patients in turkey (4). We also determined that the occupations of animal husbandry and farming were significantly associated with cchfv seropositivity . Vector ticks are generally present on the ground and on animals, which explains the risk for cchfv infection in persons who work in farming and animal husbandry . Personal protective measures such as regular examination of clothing and skin for ticks, tick removal, and use of repellents are important to prevent cchfv infection (12). We did not identify any association between seroprevalence and gender but found that cchfv seropositivity increased with age . In these regions of turkey, women contribute to farming and animal husbandry tasks and are exposed to ticks and livestock as often as men are . However, age> 40 years was significantly associated with cchfv seropositivity and reflects the age of workers in turkish agricultural areas (4,8,13). Increased cchfv seroprevalence with age exposure to blood and tissues of viremic animals during slaughter is a source of infection (12,14). However, we did not identify any association between cchfv seropositivity and contact with animals . This finding may result from a low number of viremic animals in our study region . It is known that domestic animals generally have low levels of viremia, which lasts a short time (15). However, in our study region, 79% of animals have been found to be seropositive against cchfv (4). In the study population, 89 (11.4%) persons had a history of close contact with a cchfv - infected patient . Among these 89 persons, 14 (15.7%) were seropositive, but this transmission route for cchfv was not statistically significant for our study population . However, protection against this potential transmission route is especially important for healthcare workers in hospitals that provide care to cchfv case - patients (12). This study indicated that tick exposure is the most statistically significant transmission route for cchfv in a high - risk population in turkey . Effective tick prevention aids such as tick repellents may help reduce the risk . On the other hand, the absence of cchfv seropositivity in 87.2% of the population after 4 cchfv outbreaks in turkey may suggest that this population remains at risk for infection in the future.
The effect of these agents apart from generation of desired activity must be assessed to understand the mechanism of action and side effects of the drug if any . Alzheimer is the most common cause of dementia; it is a primary degenerative disease of the brain . Onset is usually late in life with increasing impairment of memory, cognition, linguistic ability, and judgment . It is a progressive brain disorder that gradually destroys a person's ability to learn, reason, and carry out daily activities [1, 2]. The greatly reduced concentration of acetylcholine in the cerebral cortex is a significant factor in ad [3, 4]. People with alzheimer disease (ad) have lower level of acetylcholine with the development of abnormalities in cholinergic neurons . One approach to lessening the impact of these abnormalities is to inhibit the breakdown of acetylcholine (ach) by blocking the relevant enzyme ache (acetyl choline esterase) (figure 1). Che inhibitors prevent the hydrolysis of ach to choline and acetate in the synaptic clefts and result in activating cholinergic transmission . Donepezil is a piperidine - class ache inhibitor, rationally designed especially for ad [5, 6]. Donepezil was proven to improve cognitive function of mild to severe moderate ad patients and showed excellent tolerability without hepatotoxicity [79]. Donepezil is the first agent to be successfully developed specifically for the treatment of cognitive decline associated with alzheimer's disease; it is marketed under the trade name of aricept and works as an acetyl cholinesterase inhibitor . It has an oral bioavailability of 100% and easily crosses the blood - brain barrier . It is believed that donepezil works by reducing the breakdown of acetylcholine thus increasing the concentration of acetylcholine in the brain reverting it back to its normal function . Recent researches have shown that cathepsins are involved the processing of certain neuropeptides in the central nervous system (cns) where cystatin c is also present in high concentration and their concentration is suggested to play an important role in brain diseases . An imbalance between the activity of cathepsins and cystatins may lead to accumulation of potentially amyloidogenic fragments which aggregates and forms amyloid fibrils in nerve cells of ad brain in which cystatin concentration decreases . Potentially amyloidogenic fragments generated by imbalance of cathepsins and cystatin are released into the extracellular space . In normal persons, cystatins constitute a powerful regulatory system for endogenous cysteine proteinases (cathepsins) which are often secreted or leaking from the lysosomes of dying or diseased cells . They are the natural inhibitors of cysteine proteases which belongs to a super family of proteins with wide occurrence in tissues and cells . On the basis of homology, inhibition of target enzymes, and presence or absence of disulphide bonds, cystatin super family has been divided into three families . Family 1: also called as stefins include members of low molecular weight proteins (11 kda) which lack disulphide bonds and carbohydrate contents . This family includes cystatin a, b, stefins c, and stefins d. family 2: known as cystatin family represented by the inhibitors of a bit larger molecular weight proteins (13 kda) as compared to stefins and possesses disulphide bond towards the carboxyl terminal with no carbohydrates . Common example is cystatin c. kininogens or family 3 cystatins are large precursor molecules of the vasoactive kinins . They are single chain glycoproteins, which serve a variety of biological functions such as kinin delivery, induction of endogenous blood coagulation cascade, and mediation of the acute phase response . Cystatins tightly bind and inhibit the activity of cathepsins, if the activity of cathepsins is not regulated it will lead to chronic diseases . It has been probed that proteinases and their endogenous inhibitors cystatins are closely associated with senile plaque, cerebrovascular amyloid deposits, and neurofibrillary tangles in alzheimer disease . In normal person, cystatins bind to cathepsins and prevent amyloid formation; however in ad, the level of cystatin goes down and the level of cathepsins and ache enzyme increase which leads to decreases in acetylcholine and leads to formation a peptides, donepezil when supplemented it binds to ache and prevents acetylcholine breakdown thus increases the level of acetyl choline which is required for normal functioning of the brain . An earlier report showed that donepezil binds with hsa and changes its free concentration in plasma showing the possibility of conformational change in protein (figure 2). The aim was to find out if supplementation of donepezil has any effect on the activity of cystatin (major regulator of thiol proteases cathepsins b, h, and l, etc .) In the mammalian system . If the activity of these proteases is not regulated, it will lead to protease and antiprotease imbalance, a cause of several diseases . So we have investigated whether cystatin binding with donepezil has any role in the proper action of drug or leads to what kind of side effects as well as to gain knowledge about any conformational change in cystatin effecting its activity . Our study shows that donepezil unfolds cystatin which may not be able to bind to cathepsins; therefore an imbalance of protease - antiprotease occurs in the presence of donepezil leading to considerable side effect of the drug as cystatins play significant role in several diseases like arthritis cancer and cardiovascular diseases . Louis, usa). Donepezil (an alzheimer drug) was purchased from ranbaxy (india). All other reagents were of analytical grade, and double distilled water was used throughout . Buffalo brain whole mass (150 g) was brought fresh from slaughter house in an ice bucket . It was thoroughly washed with water, thin membrane and nerves were removed by forcep, and the whole brain tissue was homogenized in 50 mm sodium phosphate buffer (300 ml) of ph 7.5 containing 0.15 m nacl, 3 mm edta, and 2% n - butanol . After centrifugation at 11000 rpm for 15 minutes at 40c, residue was discarded and the supernatant was further processed . The procedure involved a combination of alkaline treatment (ph 11.0), ammonium sulphate fractionation, and gel filtration chromatography . Buffalo brain was homogenized and fractionated with ammonium sulfate between 40 and 60%; it was then dialyzed against 50 mm sodium phosphate buffer ph 7.4 containing 0.1 m nacl . Elution profile showed two protein peaks one major and one minor called as peak - i and peak - ii . Peak - i corresponding to high molecular weight buffalo brain cystatin had significant inhibitory activity and protein content; however, peak - ii with insignificant proteins concentration and low inhibitory activity was not taken into consideration for further studies . Peak - i named as bc was purified with fold purification of 384.72 and yield of 64.13% . Papain inhibitory fractions of peak - i were pooled, concentrated, and checked for purity . Brain cystatin (bc) (1 m) was incubated for 30 min with increasing concentrations of donepezil in 0.05 m sodium phosphate buffer ph 7.5 in a final reaction volume of 1 ml at room temperature . Fluorescence measurements were carried out on a shimadzu spectrofluorometer model rf-5301pc (shimadzu, japan) equipped with a 150 w xenon lamp and a slit width of 10 nm at 298 k. the fluorescence was recorded in wavelength region 300400 nm after exciting the protein at 280 nm . The uv measurement of brain cystatin in the presence and absence of drug was made in the range of 200300 nm, and the inhibitor (cystatin) concentration was fixed at 1 m while the drug concentration was varied from 0.16 m1.6 m . Absorption spectra were recorded on a double beam shimadzu uv - vis spectrophotometer uv-1700 using a cuvette of 1 cm path length . The inhibitory activity of the purified inhibitor (bc) under native conditions was assessed by its ability to inhibit caseinolytic activity of papain by the method of kunitz . The inhibitor (1 m) was incubated with increasing concentrations of donepezil at 25c for 30 min before the activity was measured . Alzheimer disease is a progressive brain disorder that gradually destroys a person's memory and ability to learn, reason, and make judgments . Ache is responsible for degradation of the neurotransmitter acetylcholine (ach) in the synaptic cleft of neuromuscular junctions and of neuronal contacts in the central nervous system [22, 23]. Cystatin (1 m) was incubated with various concentrations of donepezil varying from 2 to 10 m for 30 min . The fluorescence was recorded in the wavelength region of 300400 nm after exciting the protein solution at 280 nm for total protein fluorescence . Donepezil caused unfolding of the cystatin as indicated by enhancement in fluorescence intensity accompanied by the red shift of 40 nm as compared to max of native cystatin (340 nm) while the drug (native) shows max at 370 nm . However at 1.6 m, when it forms complex with cystatin there was shift in max of 10 nm with significant enhancement in fluorescence intensity (figure 3). Cystatin (1 m) was incubated with various concentrations of donepezil varying from 0.16 m to 1.6 m for 30 min . The fluorescence was recorded in the wavelength region 300400 nm after exciting the protein solution at 280 nm for total protein fluorescence . The path length of the sample was 1 cm in the final reaction volume of 1 ml in 0.05 m sodium phosphate buffer ph 7.5 . Cystatin concentrations were fixed at 1 m while the donepezil concentrations varied from 0.16 m to 1.6 m . Absorption spectra of native cystatin and in the presence and absence of donepezil were recorded in the range of 200300 nm . The uv absorption intensity of cystatin increased with increasing concentration of donepezil concentration; however the slight decrease in absorption intensity may be due to disruption or perturbation of absorbing groups (figure 3). Cystatin concentrations were fixed at 1 m while the donepezil concentration was varied from 0.16 m to 1.6 m . Absorption spectra of native cystatin in the presence and absence of donepezil were recorded in the range of 200300 nm in a cuvette of 1 cm path length for 30 min in the final reaction volume of 1 ml in 0.05 m sodium phosphate buffer ph 7.5 . A change in the inhibitory activity of cystatin with increasing concentration of donepezil is shown in (table 1). Effect of donepezil on cystatin function was assessed by monitoring its changes in antiproteolytic activity by caseinolytic assay of papain; 1 m of cystatin was incubated with increasing concentration of donepezil (0.161.6 m). Exposure of cystatin to increasing concentration of donepezil resulted in rapid decline of antiproteolytic activity; 85% decline in the activity was seen at 1.6 m of donepezil with more than half of the inactivation of cystatin was taking place at concentration as low as 0.32 m . Table 1 shows changes in the inhibitory activity of brain cystatin after its incubation for its inhibitory activity with increasing concentrations of donepezil . Cystatin (1 m) was treated with varying concentration of donepezil (0.16 m1.6 m) for 30 min in the final reaction volume of 1 ml in 0.05 m sodium phosphate buffer ph 7.5 . For three different sets of experiments; statistical significance was conducted employing one way anova . Drug inducing changes in protein function leading to adverse side effects is the area of continual scientific investigation [25, 26]. Even small structural differences in protein conformation can lead to drastic changes in functional parameters . Addition of small molecules such as many drugs, particularly those with local anesthetics, tranquillizers, and antidepressants, can bind to the native state and can alter the delicate balance of various interactions in proteins [2730]. Accumulation of drug molecules at certain sites in the body causes their localized high concentration leading to adverse drug reactions and ligand induced protein structure conformational changes . These are major problems complicating drug medical therapy; therefore investigations which combine the study of the conformational changes in proteins through variations of external parameters between proteins and drugs and the structure of the resulting complexes are of particular interest . These studies enable to elucidate how ligand affinity is regulated and how the protein conformation is altered upon complexation which is of crucial importance in a vast range of important biochemical phenomena . In the present work, structural and functional analysis of cystatin, a protein ubiquitously present in mammalian cells and tissues, was studied which showed a significant increase in fluorescence intensity due to unfolding of cystatin in the presence of donepezil (figure 3). Such kind of changes have also been documented earlier, after interaction of ligands (phytohormones, cytokinins, abscisic, and gibberellic acids) with wheat germ agglutinin resulting in 60% increase in fluorescence intensity of native protein . Donepezil - cystatin complexation showed 40 nm red shift in max indicating exposure of aromatic residues to the solvent caused by conformational changes in the protein [34, 35]. Absorption spectral measurements of cystatin in the presence of drug showed a peak noticeable at 275 nm and 210 nm in spectra obtained at 0.16 m donepezil (figure 4). Suggesting changes mainly due to tryptophan and tyrosine residues . There was a gradual decline in cystatin activity with increasing drug concentration resulting in 62% loss at 0.64 m donepezil (table 1). Further magnitude of decline was relatively smaller with increasing drug concentration at 1.6 m; cystatin the inhibitor retained only 15% of its antiproteolytic potential . Thus, the results indicate that the uv absorption and fluorescence emission changes in donepezil mediated interaction are due to conformational changes in cystatin mainly arising from interaction affecting the chromophoric groups of the protein which produce significant effect on the activity of cystatin . The study shows that in the presence of donepezil, cystatin gets unfolded which is a side effect of donepezil . The knowledge about the pharmacokinetics and pharmacodynamics of the drug - protein interaction continues to expand . The increased information available to clinicians might help in optimizing the use of these agents in the management of patients with alzheimer's and other diseases.
Earlier studies have reported that either caloric restriction or the inactivation of nutrient - dependent pathways is able to increase life extension in different eukaryotes . The extension of lifespan in various organisms is associated with increased resistance to oxidative stress (1). The findings of numerous studies using various models of organisms have provided indirect evidence for the hypothesis that reactive oxygen species (ros) production and subsequent induction of ros defense are essential contributors to longevity (2). As in higher eukaryotes, the unicellular yeast, saccharomyces cerevisiae, has been the most used yeast model in aging studies (3, 4). Yeast lifespan can be measured through two methods: replicative aging refers to the number of divisions a single mother cell undergoes before death, whereas chronological lifespan measures the viability of cultures at the stationary phase of the growth curve (5, 6). The fission yeast, schizosaccharomyces pombe, has been recently used as a model for chronological aging studies inasmuch as it is more similar to the last common ancestor of humans and fungi (7). Nutrient restriction increases resistance to oxidative stress, reduces macromolecular damage, and promotes lifespan in s. pombe (8 - 10). Roux et al . (11) revealed that the s. pombe gpa2 mutant exhibits not only a short lifespan but also impaired mitochondrial regulation and high production of ros . (12) reported that calorie restriction favors oxidative metabolism, ros production, and sty1 map kinase activation and this stress pathway favors lifespan extension . In s. pombe, glucose, which is a primary carbon source, is detected by g protein - coupled receptors and generates a signal via the camp - dependent protein kinase a (pka) (13, 14). The glucose sensing and signaling pathways have also been found to be involved in metabolic adaptation and cellular response to diverse stress agents (15, 16). In s. pombe, the multistep phosphorelay system and the mitogen - activated protein kinase (mapk) pathway govern the transcriptional regulation in response to oxidative stress, which is generated by the accumulation of ros (17). Mapk sty1 plays an important role in the regulation of downstream targets through triggering two transcriptional activators, atf1 and pap1, in oxidative stress response (18). These transcription factors activate or induce the expression of antioxidant genes such as ctt1 (cytoplasmic catalase), gpx1 (glutathione peroxidase), ttr1 (thioredoxin reductase), trx2 (thioredoxin), ntp1 (neutral trehalase), pgr1 (glutathione reductase), and sod1 (superoxide dismutase, sod) (19). Trehalose (, -1, 1-diglucose) is a storage disaccharide and is present in particularly high concentrations in resting and stressed yeast cells (20). Trehalose 6-phosphate (t6p) is synthesized in s. pombe from glucose 6-phosphate and udp - glucose by t6p synthetase, encoded by the tps1 gene (21), and converted to trehalose by t6p phosphatase, encoded by the tpp1gene (22). The breakdown of trehalose to glucose is catalyzed by the enzyme neutral trehalase, encoded by the ntp1 gene (23). Generally in yeast, the regulation of trehalose synthesis and breakdown is done by camp - dependent phosphorylation mechanisms (24). It has been reported that the ntp1 expression is regulated by the pathway of protein kinase cascade activated under osmotic or oxidative stress (23, 25) or by the binding of the transcription factors to elements such as camp - response element under thermal stress (26). In the present study, ird5 and ird11 were used to evaluate whether or not trehalose contributes to survival under moderate oxidative stress . In a previous study, we identified two glucose - repression - resistant mutants, namely ird5 and ird11 (27). In the ird11 mutant, the oxidative stress response is affected by glucose signaling in a manner different from that caused by glucose deprivation (28). The ird5 mutant has a lower caloric intake owing to reduced glucose consumption efficiency (29). The inefficient glucose uptake in ird5 might be a cause of increased oxidative stress response . Accordingly, in the present study, through trehalose accumulation measurement and colony - forming unit (cfu) counting, we compared the ird mutants and the wild type to clarify the possible relationship between glucose signaling, oxidative stress response, and lifespan in s. pombe . In this study, the wild type of s. pombe lindner liquefaciens (972 h) and its relative invertase mutants (i.e. Ird5 and ird11), which are resistant to glucose suppression (27), were used . The selective medium consisted of 0.5% yeast extract and 3% sucrose and 400 g / ml 2-deoxy - d - glucose (2-dog) was developed for the ird mutants . The strains were cultured in the ye medium containing 3% glucose (repressed condition) and 0.5% glucose (glucose starvation condition) and the ye medium containing 0.1% glucose plus 3% glycerol (de - repressed condition). All the chemicals were provided by sigma - aldrich, germany . The exponentially growing s. pombe cells (wild type and ird5 and ird11) under repressed conditions were split into two tubes . In the experimental group, 2 mm hydrogen peroxide (h2o2) (sigma - aldrich, germany) was added to the medium, resulting in a mild level of oxidative stress in the s. pombe cells (30). After one hour, both experimental and control cells were removed by centrifugation and washed with sterile distilled water so that their trehalose content could be measured . Chronological lifespan analyses were done on the wild type and the ird mutant cells, grown under repressed and de - repressed conditions for 21 days . Usually one day (the exponential phase) after starting the cultures, and this time point was designated as day 0, measurements were started and continued via sampling at 2-day intervals (3rd, 5th,..., 21st days of the study). This study was performed under three conditions: repressed; de - repressed; and stressed conditions . From each group cells, samples were collected on the 1st (the exponential phase), 3rd, and 5th (stationary phase) days of the study, and trehalose accumulation was measured for each case . Trehalose was extracted and assayed as is described by parrou and francois (31). The amount of the trehalose contents of the samples was measured by treatment with trehalase (sigma - aldrich, germany). The amount of the generated glucose was determined enzymatically via the glucose oxidase - peroxidase system (god - pod assay) using a commercial kit (fluitest-glu, biocon, germany). Statistical comparisons were made using the one - way analysis of variance (anova) module of graphpad prism 5 . In this study, the wild type of s. pombe lindner liquefaciens (972 h) and its relative invertase mutants (i.e. Ird5 and ird11), which are resistant to glucose suppression (27), were used . The selective medium consisted of 0.5% yeast extract and 3% sucrose and 400 g / ml 2-deoxy - d - glucose (2-dog) was developed for the ird mutants . The strains were cultured in the ye medium containing 3% glucose (repressed condition) and 0.5% glucose (glucose starvation condition) and the ye medium containing 0.1% glucose plus 3% glycerol (de - repressed condition). All the chemicals were provided by sigma - aldrich, germany . The exponentially growing s. pombe cells (wild type and ird5 and ird11) under repressed conditions were split into two tubes . In the experimental group, 2 mm hydrogen peroxide (h2o2) (sigma - aldrich, germany) was added to the medium, resulting in a mild level of oxidative stress in the s. pombe cells (30). After one hour, both experimental and control cells were removed by centrifugation and washed with sterile distilled water so that their trehalose content could be measured . Chronological lifespan analyses were done on the wild type and the ird mutant cells, grown under repressed and de - repressed conditions for 21 days . Usually one day (the exponential phase) after starting the cultures, and this time point was designated as day 0, measurements were started and continued via sampling at 2-day intervals (3rd, 5th,..., 21st days of the study). This study was performed under three conditions: repressed; de - repressed; and stressed conditions . From each group cells, samples were collected on the 1st (the exponential phase), 3rd, and 5th (stationary phase) days of the study, and trehalose accumulation was measured for each case . Trehalose was extracted and assayed as is described by parrou and francois (31). The amount of the trehalose contents of the samples was measured by treatment with trehalase (sigma - aldrich, germany). The amount of the generated glucose was determined enzymatically via the glucose oxidase - peroxidase system (god - pod assay) using a commercial kit (fluitest-glu, biocon, germany). Statistical comparisons were made using the one - way analysis of variance (anova) module of graphpad prism 5 . Of the strains under study, ird5 showed the most longevity in both repressed and de - repressed conditions . The ird11 mutant had a lower lifespan extension than the wild type under repressed condition, while it had a higher lifespan extension than the wild type under de - repressed condition . On the other hand, the lifespan extension of ird11 grown in de - repressed condition was similar to that of ird5 grown in repressed or de - repressed condition (figure 1). A, grown in repressed condition (ye-3% glucose); b, grown in de - repressed condition (ye-0.1% glucose plus 3% glycerol); (wt: wild type, r: repressed, dr: de - repressed condition). To comprehend the relationship between trehalose concentrations in the cells grown in repressed, de - repressed, and stressed conditions, the level of trehalose was estimated in the wild type and the mutant cells . The intracellular trehalose concentrations during the period of growth under repressed, de - repressed, and stressed conditions relative to the wild type and mutant cells are shown in figure 2 a, figure 2 b, and figure 2 c, respectively . The results obtained from trehalose content measurement in the three conditions were evaluated in two ways . First, each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day . Second, the trehalose content of each group was compared with that of the wild type . A, grown in repressed condition (ye-3% glucose); b, grown in repressed condition (ye-0.1% glucose plus 3% glycerol); c, grown in stressed condition (2 mm h2o2, one hour). The trehalose contents are expressed as mg glucose per 0.2 g of wet mass of the cells . Statistical significance was evaluated by one - way anova (p <0.05 - 0,001). (wt: wild type, r: repressed, dr: de - repressed, hp: oxidative stress conditions). , , * : each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day . +, + +, + + +: trehalose content of each group is compared with that of the wild type . When compared independently, as is depicted in figure 2 a, except for ird5 under repressed condition, the trehalose content was increased remarkably on the 3rd day of the study . However, a comparison with the wild type showed a sharp decrease in the trehalose level in the ird mutant . As is evident from figure 2 b, under de - repressed condition, the level of intracellular trehalose was notably increased in the stationary phase (3rd day), while no significant changes were detected in either ird5 or in ird11 . Under stressed condition (figure 2 c), during the stationary phase (3rd day), the trehalose level in ird11 was increased as a pattern similar to that observed in the wild type; nevertheless, only trace amounts of alteration were seen in ird5 . Finally, concerning the wild type during the stationary growth phase, a dramatic decrease was observed in the trehalose content in ird5 (3rd and 5th days) and ird11 (3rd day). Of the strains under study, ird5 showed the most longevity in both repressed and de - repressed conditions . The ird11 mutant had a lower lifespan extension than the wild type under repressed condition, while it had a higher lifespan extension than the wild type under de - repressed condition . On the other hand, the lifespan extension of ird11 grown in de - repressed condition was similar to that of ird5 grown in repressed or de - repressed condition (figure 1). A, grown in repressed condition (ye-3% glucose); b, grown in de - repressed condition (ye-0.1% glucose plus 3% glycerol); (wt: wild type, r: repressed, dr: de - repressed condition). To comprehend the relationship between trehalose concentrations in the cells grown in repressed, de - repressed, and stressed conditions, the level of trehalose was estimated in the wild type and the mutant cells . The intracellular trehalose concentrations during the period of growth under repressed, de - repressed, and stressed conditions relative to the wild type and mutant cells are shown in figure 2 a, figure 2 b, and figure 2 c, respectively . The results obtained from trehalose content measurement in the three conditions were evaluated in two ways . First, each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day . Second, the trehalose content of each group was compared with that of the wild type . A, grown in repressed condition (ye-3% glucose); b, grown in repressed condition (ye-0.1% glucose plus 3% glycerol); c, grown in stressed condition (2 mm h2o2, one hour). The trehalose contents are expressed as mg glucose per 0.2 g of wet mass of the cells . Statistical significance was evaluated by one - way anova (p <0.05 - 0,001). (wt: wild type, r: repressed, dr: de - repressed, hp: oxidative stress conditions). , * , * *: each group was evaluated individually and the trehalose contents of the groups on the 3rd and 5th days of the study were compared with those of the 1st day . +, + +, + + +: trehalose content of each group is compared with that of the wild type . When compared independently, as is depicted in figure 2 a, except for ird5 under repressed condition, the trehalose content was increased remarkably on the 3rd day of the study . However, a comparison with the wild type showed a sharp decrease in the trehalose level in the ird mutant . As is evident from figure 2 b, under de - repressed condition, the level of intracellular trehalose was notably increased in the stationary phase (3rd day), while no significant changes were detected in either ird5 or in ird11 . Under stressed condition (figure 2 c), during the stationary phase (3rd day), the trehalose level in ird11 was increased as a pattern similar to that observed in the wild type; nevertheless, only trace amounts of alteration were seen in ird5 . Finally, concerning the wild type during the stationary growth phase, a dramatic decrease was observed in the trehalose content in ird5 (3rd and 5th days) and ird11 (3rd day). The pka activated by glucose starvation and stress - activated mapk pathways regulate the transcription of downstream genes via protein - dna interactions at uas1 and uas2 of the fbp1 gene (32). The existence of cross - talking between sty1p and pka1p regulates not only glucose repression and oxidative stress response pathways but also trehalose accumulation . (25) reported that the expression of the ntp1 and tps1 genes in s. pombe is partially regulated by the sty1p kinase under salt - induced osmotic stress and conditions of slight oxidative stress and is fully dependent on this kinase under severe oxidative stress . Another study showed that cell viability may depend on capacity to rapidly degrade the trehalose that is accumulated during oxidative stress . The authors indicated that glutathione reductase can be inhibited by trehalose in a dose - dependent manner . On the other hand, sanchez - fresneda et al . (33) reported that the stress - induced trehalose accumulation is hog1-independent in candida albicans . The extension of lifespan in s. pombe is associated with both calorie restriction and increased resistance to oxidative stress (10 - 12). (34), our results suggest that lifespan extension in s. pombe may not be associated with oxidative stress resistance and trehalose accumulation . The oxidative stress - resistant mutant ird11, which is affected by glucose signaling in a manner different from that caused by glucose deprivation (28), had a lower lifespan extension than the wild type under repressed condition and had a higher lifespan extension than the wild type under de - repressed condition . Indeed, the lifespan extension of ird11 grown in de - repressed condition was similar to that of ird5 grown in repressed or de - repressed condition . We suggest that the longevity of the ird5 mutant grown in both repressed and de - repressed conditions might be, in part, in consequence of glucose depletion condition caused by a reduced glucose consumption rate in these cells compared to the ird11 mutant and the wild type . Because, contrary to ird11, there is an adaptive response to oxidative stress in ird5, caused by glucose sensing / signaling which emerges in glucose starvation (29). In light of the results of the present study, it seems that lifespan extension is mostly related to glucose sensing / signaling rather than oxidative stress response and trehalose accumulation.
Bovine ubiquitin (8.56 kda, 76 amino acids [aa]) and cytochrome c (12.22 kda, 104 aa), and equine apo - myoglobin (16.95 kda, 153 aa) were purchased from sigma aldrich (poole, uk) and used without further purification . Methanol (fisher scientific, leicestershire, uk), water (j. t. baker, deventer, netherlands), and formic acid (fisher scientific) were used for preparing the electrospray solution . Peptide substance p (sigma aldrich) was used to calibrate the instrument and for comparison with fragmentation of the intact proteins . Protein samples (13 m in 49.5/49.5% of h2o / ch3oh and 1% of formic acid) were directly infused via an external nanospray ionization source (advion biosciences, ithaca, ny) into a 7 t thermo finnigan ltq ft mass spectrometer (thermo fisher scientific, bremen, germany). Automatic gain control (agc) was used to accumulate precursor cations in the ion trap (target 1 10, maximum fill time 2 s) before transporting them into the icr cell with a trapping voltage of 1 v. ir excitation was carried out in the icr cell using a 75 w in - built co2 laser (synrad, mukilteo, wa). Ir fluency was controlled via thermo software and measured as percent of the maximum (75 w). Electrons for ecd were produced by an indirectly heated barium - tungsten cylindrical dispenser cathode (5.1 mm diameter, 154 mm from the cell, 1 mm off axis) (heat - wave labs, watsonville, ca). The current across the electrode was 1.1 a. it is important for the matter of this paper that in our instrument the ir beam crosses the icr cell at angle in respect to its axis, whilst the electron beam is aligned to be parallel to the axis of the icr cell . Raw ms data were analyzed by use of xcalibur 2.05 software (thermo fisher scientific), where the xtract program was used for calculating monoisotopic masses (44% fit factor, 25% remainder). Prosight ptm (https://prosightptm.scs.uiuc.edu) was used to search for c, z and b, y protein fragment ions . The mass accuracy for the search the lists of masses from ecd ms / ms spectra were searched both for standard c, z ions, and for hydrogen transfer c, z fragments . Tsybin et al . Observed modulation of ecd by imm for substance p and ubiquitin 9 + ions in a homebuilt 9.4 t esi - q - ft - icr mass spectrometer . We have observed similar ecd modulation in our thermo finnigan ltq ft instrument, and, in addition, modulation of irmpd and infrared - ai ecd efficiencies with the same periodicity . Data for ecd and irmpd of substance p are shown in figure 1 . Fragmentation efficiencies were calculated as the ratio of the sum of all fragment ions abundances to the 2 + precursor ion abundance . Time zero in figure 1 corresponds to a delay of 44.73 ms between ion injection into the icr cell and the first maximum of ecd efficiency . That time delay was set automatically during program - controlled calibration of ecd and was not changed thereafter . An irradiation time of 70 ms is typically used for ecd of peptides, because longer irradiation results in secondary, neutralizing electron capture and leads to a decrease in the number of ion fragments and, therefore, ecd efficiency (figure 1). To synchronize the ecd and irmpd delays, all our irmpd measurements for substance p and the proteins were carried out with both irmpd and ecd options activated in the controlling software, ecd energy and delay both zeroed, and ecd duration 0.03 ms, the minimum allowed by the software . Our irmpd data for substance p and varied irmpd delay show modulation with the same periodicity as in the ecd data . The width of irmpd peaks (full width at half maximum peak height) is less than that of ecd peaks because of the shorter duration of irmpd (20 ms versus 70 ms for ecd). 30 to 40 ms towards the beginning of the timescale with respect to the maxima of ecd efficiency (figure 1). The offset between the maxima of the irmpd and ecd peaks can be explained by the off - axis position of the ir laser beam in the instrument . A schematic representation of the geometrical arrangement of electron and ir laser beams in the icr cell (55 mm diameter) is given in figure 2 . As described above, the electron beam is positioned slightly off - axis and aligned parallel to the cell axis . Additionally, the ir beam is deliberately aligned at an angle to the icr cell axis and crosses the ion trajectories in a place different from their intersection with the electron beam . As the ion cloud moves along the imm trajectory (counter clockwise in figure 2, top), first it crosses the ir beam and then the electron beam . Time zero, therefore, corresponds to the first intersection with the electron beam, 44.73 ms after the ions are trapped in the icr cell . 85 ms period and 30 ms offset between their maxima was observed for ubiquitin, cytochrome c and myoglobin . The results for ecd and irmpd of 6 + ions of ubiquitin are shown in figure 3 . The relative intensity of the reduced (m + nh) ions was used as a measure of electron capture efficiency (figure 3a). Irmpd depletion of the precursor ion peak was used as a measure for irmpd efficiency (figure 3b). Depletion measurements allow a fast test of the impact of imm modulation on both ecd and irmpd, and fast in situ optimization of the time sequence for activated ion ecd of protein ions, as only 10 microscans (transients) are sufficient for each data point . A more appropriate way of calculating absolute ecd and irmpd efficiencies involves summing the intensities of all fragments, as performed for substance p (figure 1). That approach is not suitable for in situ optimization: a much larger number of microscans (transients) would have to be accumulated to provide good signal - to - noise ratio for ecd fragments of intact proteins, and the data analysis would be lengthy (ecd of proteins results in many fragments). The positions of both the ecd and irmpd maxima coincide for substance p and ubiquitin, (figures 1 and 3). The positions of the maxima and the modulation period (85 ms) also remain the same for cytochrome c and myoglobin (data not shown). Ergo, the modulation does not depend on ion m / z ratio, as is expected for ion magnetron motion [32, 33]. Due to the geometry of the ir alignment, most of the interaction between the ir beam and the ion population takes place near the middle of the icr cell (figure 2). The ions are fast moving in and out of this region because of the z - trapping motion . Therefore our results in figures 1 and 3 represent an average over many periods of z - trapping motion . The implication of the above findings for implementation of ai ecd on this and similar types of instrument, particularly for protein analysis, is that proper synchronization of ion activation and ecd with the ion magnetron motion must be ensured . It is not possible to carry out ecd and ir activation simultaneously, because the maxima for ir activation do not overlap with those for ecd . Ir activation has to be carried out either before or after the ecd event and synchronized with imm . As mentioned above, effective activation (both pre- and post - ecd) leads to depletion of charge - reduced ions and appearance of new ecd fragments in the ms / ms spectra . Thus depletion of the charge - reduced ions can be used as a measure of activation efficiency . Time - dependent variation of the depletion of reduced (m + 6h) ions under post - ecd activation is presented in figure 3c . Electrons were injected for 10 ms at time zero for each data point, and the delay for ir irradiation was varied . The data in figure 3c demonstrate that imm also modulates the efficiency of ai ecd, and the efficiency maxima for post - ecd activation (figure 3c) overlap with those for irmpd (figure 3b). Ir irradiation (70 ms duration) overlaps the first irmpd efficiency maximum (figure 3b), and the electron irradiation is carried out after that at the times corresponding to maximum ecd efficiency (figure 3a). In general, optimization of activated ion ecd was achieved as follows . First, ecd duration and ecd cathode voltage were tuned to obtain the maximum number of different ecd fragments from the isolated charge state . Ecd delay was set at zero, and ir activation was not used in this step . In cases where only a few, or no, fragments could be produced, thus precluding tuning, optimized values for the next highest charge state were used . Typically, the optimized ecd duration was 5 to 10 ms and the ecd cathode voltage was between 2 and 3.5 v. second, ecd time delay was chosen either for pre - ecd or post - ecd activation . With the exception of the kinetic studies (see below), the time for ecd was set at t = 0 (corresponding to the first ecd efficiency maximum) for post - ecd activation; and t = 85 ms (corresponding to the second ecd maximum) for pre - ecd activation . Third, the time for ir activation was centered at t = 60 ms (corresponding to the first irmpd efficiency maximum, figure 2b) for both pre- and post - activation . The duration of ir irradiation was usually in the region 20 to 120 ms, but extension of ir activation over several irmpd efficiency maxima was also attempted . Fourth, ai ecd ms / ms spectra for different values of ir fluency were acquired . The mass spectra obtained were analyzed and the number of fragments identified by prosight ptm was plotted versus ir fluency . 7 + ir activation leads to the appearance of fragments from new cleavage sites . Figure 4 shows the numbers of different fragment ions from ubiquitin 6 + and 7 + charge states . For pre - ecd activation (figure 4a and b) the duration of ir irradiation was 85 ms, and ecd duration and delay we found that these settings provided a greater (15%) total number of fragments than opening a shorter, 30 ms window for ir activation exactly during the first irmpd efficiency maximum . This improvement could be due to the heating of the icr cell walls by the ir beam between irmpd maxima followed by black - body irradiation of the trapped ions from the cell walls . Increased ecd fragmentation in a heated icr cell was reported by mclafferty and coworkers [20, 27, 28]. For post - ecd activation, ecd duration was 10 ms, and ir irradiation delay and duration were 30 and 100 ms, respectively . Ubiquitin 6 + ions demonstrated poor fragmentation without ir activation: no more than 18 different c and z fragment ions could be produced . Increasing the ir fluency to 30% of the maximum led to an increase in the number of different c and z ions to 50 . This was accompanied by a significant increase in the number of z fragments, and a smaller increase in y and c fragments . The effect was stronger for pre - ecd activation than for post - ecd activation, with a total of 50 and 41 bonds cleaved, respectively . 25% increase in the total number of ecd fragments (figure 4b and d). In all four cases, fluency above 30% to 35% of the maximum the number of c and z fragments diminishes rapidly, and is accompanied by an increase in the number of different b and y fragments . These threshold values correspond to the onset of irmpd of (m + 6h) and (m + 7h) ions . In all the above cases, c and z ions constitute a significant proportion of the total fragment yield . Figure 5 shows summaries of the fragmentation of ubiquitin 6 + ions for different conditions of ir activation and ecd . Without activation most of the ecd fragments come from the region val5leu15 and the two terminal residues (figure 5a). Ir activation results in fragments from other regions of the protein, (figure 5b d). The z ions originate mostly from the region of residues 4871 for both pre- and post - ecd activation . In the cases of short, 25 ms, pre - ecd activation and long, 100 ms, post - ecd activation, this region of the molecule is underrepresented by standard c and z ions, and searching for z ions is essential for achieving maximum sequence coverage (figure 5c, d). The results for the 7 +, 8 +, and 9 + ions of cytochrome c and the 11 +, 12 +, and 13 + ions of myoglobin are presented in tables 1 and 2 . As for ubiquitin, the higher charge states of these two proteins demonstrate extensive bond cleavage under ecd, and ir activation does not increase the number of cleaved bonds . Ecd of the lower charge states benefits from ir activation, but the total number of ai ecd fragments decreases with decreasing charge state for z + 9 in the case of cytochrome c (table 1), and z + 12 in the case of myoglobin (table 2). Optimal ir fluencies range from 20% to 40% depending on the method of activation, protein and its charge state . The threshold to irmpd of the precursor ions varies between 35% and 40% (supplemental figures 1 and 2, which can be found in the electronic version of this article). As has been shown previously, (ai) ecd fragments were not observed from around the region cys14-cys17of cytochrome c ions, i.e., where the heme group is covalently bound to the protein, (supplemental figure 3) [20, 34]. The numbers of c and z fragments produced under ir - ai ecd of cytochrome c is larger than that of c and z fragments for all three ions (table 1). In contrast to ubiquitin, there are more c ions than z ions (supplemental figure 3). A number of c and z fragments were also produced by ai ecd of myoglobin (table 2), though their contribution to the total fragment yield is much smaller than in the case of cytochrome c. neither of the two proteins unfolds completely with ir activation, e.g., the pro100-gly121 region of the myoglobin 13 + ion remains underrepresented by ai ecd fragments (supplemental figure 4). Pre - ecd activation produces a larger overall number of fragments for all three cytochrome c charge states, although the increase is marginal for the 9 + charge state . For myoglobin, post - ecd activation produces more fragment ions for the 11 + ion (table 2). The decrease in ecd efficiency of protein ions with decreasing charge state has been well documented in the literature [20, 2730, 35], and is explained by a decrease in coulomb repulsion energy available for repelling complementary protein fragments and simultaneous increase in the number of noncovalent bonds holding the fragments together . Our data indicate that there are also limitations on protein unfolding by ir activation, which depend on the m / z value of the protein ion . It was not possible to cleave more than 10% of the backbone bonds for charge states below 5 + for ubiquitin, 7 + for cytochrome c, and 10 + for myoglobin, i.e., for charge states with less than one proton per 15 amino acids . For those charge states for which ecd fragmentation benefits from ir activation, the optimum ir fluency is 20% to 40% of the maximum, i.e., 50% to 100% of the threshold to irmpd of the precursor ions . In most cases, pre - ecd activation resulted in higher efficiency in terms of the number of n c bonds cleaved . Increasing the duration of ir activation to overlap several irmpd efficiency maxima the only result of such increase was lowering the ir fluency at which irmpd of the precursor ion started . That result is expected for a threshold fragmentation technique, such as irmpd, where the threshold to fragmentation can be reached either in high - heat regime over a short period of time, or in low - heat regime over a longer period . The results show that the ratio of the number of c and z fragments to that of c and z fragments is larger for post - ecd activation than for pre - ecd activation . That observation can be explained by the different fragmentation mechanisms of these two methods . In pre - ecd dissociation, the even - electron precursor ion is first unfolded by ir activation, and then subjected to ecd . Ecd fragments are released almost immediately after electron capture, and there exists very little time for h transfer between c and z fragments . In post - ecd activation, charge - reduced noncovalent complexes of ecd fragments are formed, and after some delay, dissociated by ir activation . Survival of the radical complex facilitates h transfer and the production of c and z ions . The time intervals for ecd or irmpd were set at each of the subsequent ecd or irmpd maxima . Durations were constant for all measurement points (10 ms for ecd and 25 ms for irmpd). Both the electron capture and irmpd efficiencies decrease with time at approximately the same rate . Exponential fits for the curves in figure 5a give decay times of 765 165 ms for the ecd data and 740 140 ms for the irmpd data . These values are very similar indicating that the effect causing the decrease in efficiencies is the same for ecd and irmpd . That effect is most probably diffusion of the ion cloud in the icr cell caused by coulombic repulsion between the trapped cations . As the ion cloud spreads in space and time, fewer ions are subjected to electron or ir irradiation . As described above, depletion of the charge - reduced ions in ecd mass spectra can be used as a measure of overall protein unfolding following ir activation . In pre - ecd activation, if the time delay between ir activation and ecd is increased, more time exists for the protein to refold before electron capture . The newly (re)formed noncovalent bonds should prevent ecd fragments from separating, leading to an increase in intensity of the charge - reduced ions . Thus measuring depletion of the reduced ions for different delays between the ir activation and the ecd event can be used for monitoring protein refolding in the gas - phase . To account for the decrease in electron capture efficiency caused by ion cloud diffusion, the intensities of charge - reduced ions with and without ir activation must be measured at the same ecd delay . Using slow ecd (1.2 s duration), mclafferty and coworkers demonstrated that the 7 + ions of ubiquitin refold after ir excitation in 1 to 2 s . However, in our instrument, these ions fragment extensively in 10 ms ecd indicating that they are unfolded . We, therefore, used ubiquitin (m + 6h) ions in our kinetic measurements . Ir activation was centered at the first irmpd efficiency maximum (figure 3b) with ir fluency at 30% . The time points for ecd were set at each of the subsequent ecd efficiency maxima (figure 3a). The depletion of the (m+6h) reduced ion drops from 93 to 13% over a period of 1.2 s, which is close to the refolding time of the 7 + ion found by mclafferty and coworkers . The depletion of the (m + 6h) reduced ion decreases with ecd delay at the same rate . The comparable behavior of the (m + 6h) and (m + 6h) ions is because their depletion in ecd spectra is affected by refolding of the same (m + 6h) precursor ions . For ecd delays greater than 1 s, ai ecd fragments are formed mostly from the same segments of the molecule as for ecd without activation, (figure 5e). That observation suggests that the ubiquitin 6 + ion folds back to the same, or similar, conformation as it had before the activation . As discussed above, post - ecd activation probes the structure of the backbone - cleaved radical complex held together by noncovalent interactions . The efficiency by which the fragments are released from the complex is affected by the time delay between ecd and ir activation only if new covalent bonds form between the backbone fragments within the charge- reduced ion . Formation of such bonds in the complex should be possible because the complex contains radical z or c ions . New covalent bonds cannot be destroyed by ir activation when it is carried out below the threshold to irmpd . Data for depletion of (m + 6h) and (m + 6h) ions by post - ecd ir activation for different ecd - ir delays are shown in figure 6b . Ir activation duration was 70 ms and coincided with the subsequent irmpd efficiency maxima (figure 3b). The depletion for each ecd delay was measured against the intensities of charge - reduced ions without activation for zero ecd delay, i.e., the effect of ion diffusion should be reflected in the measurements . The depletion of charge - reduced ions (figure 6b) does decrease with increasing ecd - ir delay . However, the rate of depletion of (m+6h) ions is less than that observed in the irmpd of (m+6h) ions, (figure 6a). The difference in the diffusion rate can be explained by a smaller charge on the (m+6h) ions c.f . The force of coulomb repulsion between ions is proportional to the square of the charge of the ion, therefore ions in the higher 6 + charge state experience stronger repulsion and their diffusion should happen faster than that of the ions with 5 + charges . Low rate of decrease in the depletion of (m + 6h) ions with the ir activation delay indicates that there is no new chemical bond formation in these radical ions . In the case of doubly - charge - reduced (m + 6h) ions, the rate of depletion is much greater than that for both (m + 6h) and (m + 6h) ions, i.e., occurs faster than diffusion . The increasing stability of the (m + 6h) ion may be an indication of the formation of new covalent bonds within this biradical cationic complex between the ecd event and ir activation . Our results are in accordance with those of kleinnijenhuis et al ., who reported similar recombination of two unpaired electrons and formation of new chemical bonds in cationic biradicals of lacticin 481 following double electron capture . In our study for substance p, ubiquitin, cytochrome c, and myoglobin the period of ion magnetron motion was ca . 85 ms for 1 v trapping voltage in the icr cell of a thermo finnigan ltq ft mass spectrometer . Periodic modulation of ir activation and ecd by ion magnetron motion should be the same for other proteins because of its invariance in respect to ion m / z value . Either pre - ecd or post - ecd ir activation can be employed for those protein ions, which require additional vibrational excitation to destroy their internal noncovalent bonds . These two methods of activation are physically different in respect to the ions they affect, and demonstrate different kinetics . However both methods require precise synchronization of both ecd event and ir activation with the ion magnetron motion . The optimum fluency for ir activation depends on the nature of the protein and its charge state . In our study 50% to 100% of the value for the threshold of irmpd of the precursor (m + nh) ion . In the search for fragments, c and z ions should not be overlooked, as they may contribute significantly to the total number of fragments and also bear information from those segments of the protein, which are underrepresented by the other types of ion fragments . Kinetic experiments on proteins in the icr cell on a time scale larger than imm period should be performed only after careful selection of the time windows in respect to the ion magnetron motion . Kinetic information must also be deconvoluted from the diffusion of ion cloud in the cell.
There is an awareness for researchers of the need to share experimental data using the internet . Proteomics, which is the study of the entire protein complement expressed by a genome in a cell, holds a key position in the present biology (1). For the separation of such complex protein mixtures, 2-dimensional electrophoresis (2-de) the proteins are detected by chromophoric staining and appear as colored spots on the 2-d gels that can be scanned for computerized spot detection . Afterwards, to determine protein content, spots are analyzed with mass spectrometry either directly or after in - gel enzymatic cleavage for peptide mass fingerprinting . Mass spectrometry results are compared with theoretical data from protein databanks allowing content identification of protein spots . Thus, researchers desirous of sharing their experimental 2-de data need to display: 2-de experimental conditions, gel pictures with stained and numbered spots and, for a given spot, the protein(s) content with appropriate links to the identification pages in databanks . When considering the high and always increasing amount of results obtained from a single 2-de gel (commonly up to 1,000 spots), the more convenient way for storing, organizing, and displaying them lies undoubtedly in internet databases . Php, a recursive acronym that stands for php: hypertext preprocessor, is a widely used general - purpose html - embedded scripting language that especially suits for web development . The code is executed on the server side and allows to dynamically generate pages, in particular, using information accessed from databases . Now, most of the web hosting companies offer the opportunity to use mysql databases and dynamic languages such as php . Thus, php could facilitate 2-de presentations on web sites because information can be stored in a database and assembled using php commands into web pages on the fly when requested . It also allows to create user - friendly interfaces to store easily content into the database tables . We have developed a php - written module, namely phproteomicdb, that can be used in the purpose to share 2-de data without knowledge for php or database manipulations . It can display smartly online 2-de gel data, including gel characteristics, gel pictures, and numbered spots with their related identifications pointing to their reference pages in protein databanks (swiss - prot or ncbi). Using phproteomicdb implies that your web site hosting company must have php - mysql enabled server, which is usually the case . No technical or php knowledge is required except a few basics about web site management . Download the last compressed version of the module from http://www.huvec.com/index.php3?rub=download and unzip the downloaded file on your local drive to obtain: (1) a folder (phproteomicdb), (2) a file for data table creation (proteomicdb.sql), and (3) a readme.pdf file for ultimate detailed instructions . 2 . Upload the phproteomicdb folder to your web site root directory using any ftp software, after having updated the mysql database configuration file with your own parameters (login, password,). 4 . Execute the proteomicdb.sql file using the phpmyadmin interface of your mysql database to create the data tables to store your 2-de gel data . Fill these tables with data of your gels, spots, and proteins in the user - friendly administration area using forms and drop - down menus upload your pictures and documents (gel pictures in jpg format, pdf documents,) in the appropriate folders of the module . When achieved, click the link installed on your web site at step 3 to display the module index page with pictures and characteristics of the 2-de gels . When clicking one of these pictures, the list of the spots and the link with the attached pdf document related to this gel are displayed . Finally, clicking a spot number allows to access a new page that displays the identified proteins in this spot (figure 1) with hypertext links to their reference pages in the protein databanks (swiss - prot or ncbi). If necessary, specific pages in the administration area allow canceling or updating data . For every page generated with the module, the retrieved data are merged with page templates that provide minimal components controlling the style of presentation . Thus, to respect his / her web site graphic chart, the webmaster is free to edit the php pages displaying his / her data (index.php3, 2dpatterngel.php3, and identification.php3) to add html codes for style or decoration . As an example what distinguishes our work from others is that it allows researchers that are newcomers in web site management to benefit from the power and the flexibility of the php dynamic language and the efficient use of databases . For people who are reluctant to use complex tools such as the make 2d - db ii package (2), which is a federated database for 2-de users, the development of the module is being pursued with future updates with the latest version available on our web site at http://www.huvec.com/index.php3?rub=download.
Nasal septal perforation (nsp) is a rare disorder characterized by composite loss of mucosa and bone or cartilage that compose the nasal septum . Septal surgery, trauma, inflammatory diseases, and use of topical nasal sprays have been implicated in the etiology of nsp (2, 3). Although most of the patients with nsp are asymptomatic, complaints may occur, depending on the size and location of the perforation . Patients with posterior septal perforations are usually asymptomatic . Anteriorly located small perforations may cause a whistling sound while breathing, and patients with larger anterior perforations complain of dry and blocked nose, crusting, foreign body sensation, and bleeding . Nasal septal perforations may also alter nasal airflow patterns and physiology . Although a number of studies have evaluated the effect of nasal septal deviation and its surgical treatment on sleep - disordered breathing (sdb), there is little data on sleep parameters and sdb in a cohort of patients with nasal septal deviation . Similarly, to the best of our knowledge, no studies in the english literature have investigated the effect of nsp and its treatment on polysomnographic parameters . Therefore, in this study we aimed to investigate polysomnographic sleep and respiratory parameters in patients with nsp as well as changes in those parameters after treatment of nsp . To the best of our knowledge, we conducted a clinical study at the otorhinolaryngology department of ankara numune training and research hospital . All investigations were performed in accordance with the declaration of helsinki on biomedical studies involving human subjects, and informed consent was obtained from all participants before the study began . The participants were selected from patients who presented with nsp and were admitted to the otorhinolaryngology clinic . Prior to the onset of the sleep study, the data were registered for each participant individually, including age, sex, height and weight (to calculate body mass index [bmi]), medical history of comorbid diseases, previous surgeries, and complaints caused by nsp . Exclusion criteria included nasal problems other than septal perforation (e.g., nasal polyps, chronic rhinosinusitis, allergic rhinitis, and septal deviation), other sleep disorders (e.g., insomnia, hypersomnia, and sleep - related movement disorders), and neurologic and psychiatric disorders . First, all patients had a detailed otorhinolaryngologic examination, and the location of nsp was determined by diagnostic nasal endoscopy . The septal perforation width and length were measured in millimeters and septal perforation area was calculated as width length . After detailed medical history - taking and physical examination, participants who were found to be appropriate to include in this study completed the epworth sleepiness scale (ess) questionnaire and underwent a full - night polysomnography (psg) study . The ess is an 8-item questionnaire designed to capture an individual s propensity to fall asleep during commonly encountered situations, on a scale ranging from 0 to 3 . The scores for the 8 questions are added together to obtain a single total score that can range from 0 to 24 . In adults, we used the alice 5 psg (alice 5 diagnostic sleep system, philips respironics, philips healthcare, the netherlands) device to record baseline and post - procedure psg . All psgs were performed during at least 7 hours of spontaneous sleep, under the supervision of an experienced sleep technician in the sleep center of ankara numune education and research hospital . The channels included in psg were 4-channel electroencephalography, 3-channel electromyography (mentalis, and right and left tibialis anterior muscles), 2-channel electrooculography (right and left eyes), 2-channel electrocardiography, nasal airflow, thoracic and abdominal respiratory movements, pulse oximetry, and body position . Sleep and respiratory parameters were evaluated according to the american academy of sleep medicine (aasm) criteria, consisting of non - rapid eye movement (nrem) stages n1, n2, n3, and rem stage during sleep . Stage 2 is identified by the presence of k - complexes or sleep spindles (or both). Stage 3 (n3) represents the deepest stages of sleep and is also described as slow - wave sleep . Rapid eye movement (rem) sleep is commonly described as paradoxical sleep and is characterized by a highly activated brain within a paralyzed body . Rem sleep is identified by the combination of 3 conditions: (1) the eeg returns to a relatively low - voltage, mixed - frequency pattern within cessation of sleep spindles, k - complexes, and high - amplitude slow waves; (2) the chin emg falls to the lowest level of the recording; and (3) the eog channels demonstrate the presence of rapid eye movements . Psg records were scored according to the guidelines american academy of sleep medicine manual for scoring of sleep and associated events, version 2.0 (2014). Following acquisition of baseline psg data, a silicone septal button was placed under local anesthesia to cover the perforation, and complete closure of the perforation was confirmed by endoscopic nasal examination . A post - procedure psg was obtained at least 7 days after the insertion of the septal button . Psg parameters, including sleep efficiency, durations of stages 1, 2, and 3 and rem, total apnea hypopnea index (ahi), rem ahi, non - rem ahi, and mean and lowest po2 saturation were compared between the baseline and post - procedure recordings . Baseline and post - procedure parameters related to sleep were also compared after dividing the patients into 2 groups, those with ahi <5 and those with ahi 5 . Statistical analysis was performed using spss version 11.5 for windows (spss, inc ., the paired samples t test was used for the variables with a normal distribution, and wilcoxon matched pair test was used for variables that were not normally distributed . The independent samples t test was used for the variables with normal distribution in relation with the perforation size, and the mann - whitney u test was used for the variables that were not normally distributed . Normally distributed variables are presented as mean sd, whereas variables that were not normally distributed are shown as median [interquartile range (iqr)]. The ess is an 8-item questionnaire designed to capture an individual s propensity to fall asleep during commonly encountered situations, on a scale ranging from 0 to 3 . The scores for the 8 questions are added together to obtain a single total score that can range from 0 to 24 . In adults, an ess score 10 indicates increased daytime sleepiness . We used the alice 5 psg (alice 5 diagnostic sleep system, philips respironics, philips healthcare, the netherlands) device to record baseline and post - procedure psg . All psgs were performed during at least 7 hours of spontaneous sleep, under the supervision of an experienced sleep technician in the sleep center of ankara numune education and research hospital . The channels included in psg were 4-channel electroencephalography, 3-channel electromyography (mentalis, and right and left tibialis anterior muscles), 2-channel electrooculography (right and left eyes), 2-channel electrocardiography, nasal airflow, thoracic and abdominal respiratory movements, pulse oximetry, and body position . Sleep and respiratory parameters were evaluated according to the american academy of sleep medicine (aasm) criteria, consisting of non - rapid eye movement (nrem) stages n1, n2, n3, and rem stage during sleep . Stage 2 is identified by the presence of k - complexes or sleep spindles (or both). Stage 3 (n3) represents the deepest stages of sleep and is also described as slow - wave sleep . Rapid eye movement (rem) sleep is commonly described as paradoxical sleep and is characterized by a highly activated brain within a paralyzed body . Rem sleep is identified by the combination of 3 conditions: (1) the eeg returns to a relatively low - voltage, mixed - frequency pattern within cessation of sleep spindles, k - complexes, and high - amplitude slow waves; (2) the chin emg falls to the lowest level of the recording; and (3) the eog channels demonstrate the presence of rapid eye movements . Psg records were scored according to the guidelines american academy of sleep medicine manual for scoring of sleep and associated events, version 2.0 (2014). Following acquisition of baseline psg data, a silicone septal button was placed under local anesthesia to cover the perforation, and complete closure of the perforation was confirmed by endoscopic nasal examination . A post - procedure psg was obtained at least 7 days after the insertion of the septal button . Psg parameters, including sleep efficiency, durations of stages 1, 2, and 3 and rem, total apnea hypopnea index (ahi), rem ahi, non - rem ahi, and mean and lowest po2 saturation were compared between the baseline and post - procedure recordings . Baseline and post - procedure parameters related to sleep were also compared after dividing the patients into 2 groups, those with ahi <5 and those with ahi 5 . Statistical analysis was performed using spss version 11.5 for windows (spss, inc ., the paired samples t test was used for the variables with a normal distribution, and wilcoxon matched pair test was used for variables that were not normally distributed . The independent samples t test was used for the variables with normal distribution in relation with the perforation size, and the mann - whitney u test was used for the variables that were not normally distributed . Normally distributed variables are presented as mean sd, whereas variables that were not normally distributed are shown as median [interquartile range (iqr)]. Nineteen patients diagnosed with nsp between september 2013 and april 2014 were included in the study . The mean age of the patients was 42.2613.20 years (range, 2365 years). Among the total of 19 patients, 15 (79%) patients had septoplasty, 2 (11%) had use of nasal sprays, 1 (5%) had nasal trauma, and 1 (5%) patient did not have any clear etiologic factors in the history . The most common complaint was dry nose and crusting (n=19, 100%), followed by nasal blockage (n=13, 68%), whistling during respiration (n=5, 26%), and daytime sleepiness (n=2, 10%). The mean body mass index (bmi) was 27 (range, 21.4434.53), and the mean ess score was 4 . When baseline psg parameters were analyzed, it was seen that sleep efficiency and percentages of n1, n2, and n3 were within normal limits; however, there was a small decrease in percentage of rem sleep (15.45.2,% total sleep time) (table 1). Rem sleep accounts for about 2025% of the sleep time in normal adults (9) and in our cases, percentage of rem sleep was less than in normal adults . Baseline ahi was <5 in 9 patients, 514 in 5 patients, and 15 in 5 patients . Median baseline ahi was 5.30 (14.40), and there was only a small deviation from the normal limits . On the other hand, median supine ahi was 10.00 (42.10), and there was a tendency for supine position - dependent sleep apnea in patients with nsp . Median ahi [5.30 (14.40) vs. 2.40 (14.50)], and median supine ahi [10.00 (42.10) vs. 6.60 (37.00)] decreased after correction of the perforation, but the differences were not statistically significant . Other studied baseline psg parameters did not change significantly after treatment of nsp (table 1). The median nsp size was 66.00 (86.00) mm2 (range, 5402 mm). Ten (53%) patients had perforations 66 mm, and 9 (47%) patients had perforations> 66 mm . We divided the patients into 2 groups: ones with a perforation size 66 mm, and the ones with a perforation> 66 mm . None of the studied parameters showed significant differences between the groups (p>0.05 for all). However, the median baseline supine ahi was 10.10 (34.15) in the group with a perforation> 66 mm, and it was 8.55 (51.43) in the group with a perforation size 66 mm . Although median supine ahi decreased to 1.60 (28.30) after closure of the perforation in the group with a perforation> 66 mm, the difference was not statistically significant (p=0.130). There were no significant changes in the 2 groups for other baseline and post- procedure parameters studied (p>0.05 for all) (table 2). The patients were divided into 2 groups according to their ahi: ones with ahi <5, and ones with ahi 5 . None of the parameters studied in the 2 groups showed significant changes after closure of the perforation (table 3). Factors relating to anxiety and stress are the most important concomitants of sleep complaints in the general population . The nasal airway is quite rigid during sleep, and, in contrast to the oropharyngeal segment, it does not collapse . A number of studies investigated the presence of nasal septal deviation and the impact of its surgical correction on sleep quality and respiratory parameters in patients with obstructive sleep apnea, showing that nasal surgery alone was not effective in restoring a normal sleep architecture or in treatment of obstructive sleep apnea [1114]. However, sufiolu et al . Reported that nasal surgery might lead to a reduction in continuous positive airway pressure (cpap) levels . On the other hand, sleep disorders in patients with nasal septal deviation were investigated in only a few studies . Increased airway resistance, unstable mouth breathing, and nasal reflexes may cause sdb or exacerbate those that already exist . The first study published on nasal airflow in nsp was conducted by grtzenmacher et al ., and reported that there was not a correlation between nsp size and abnormal aerodynamics . Air temperature in patients with nsp was significantly lower than the controls with normal noses, due to air mixing in nsp . The airflow passing through the perforation was turbulent and fast, and airflow speed was higher towards the rear end of the perforation . In addition, blood flow was greater at the edges of the perforation . Steady turbulent flow occurring at the sagittal plane caused prolonged and extensive mucosal contact, leading to mucosal irritation and dryness . Nsp may cause nasal obstruction and lead to sleep problems, similar to nasal septal deviation . Our study is the first one in the literature that studied polysomnographic sleep parameters in patients with nsp . The results of this study indicate that rem sleep duration was shorter in patients with nsp, and it did not increase after treatment [15.45.2 vs. 16.44.2]. Rem sleep accounts for about 2025% of the sleep time in normal adults (9) and in our cases, percentage of rem sleep was less than in normal adults . In our study, in 5 patients ahi values were 15 . Therefore, in these patients, obstructive sleep apnea may cause lower rem percentages . In addition, both median ahi [5.30 (14.40) vs. 2.40 (14.50)], and median supine ahi [10.00 (42.10) vs. 6.60 (37.00)] decreased after correction of the perforation . There was a great reduction in median supine ahi in patients with a perforation size> 66 mm [10.10 (34.15) vs. 1.60 (28.30)]. Second, we analyzed only objective parameters, and not the subjective ones after closure of the perforation . Third, we treated nsp with a prosthesis (silicone nasal septal button), and not with surgery . Our preliminary results indicated that nsp did not cause any deterioration in objective sleep parameters as determined by psg, other than a decrease in rem sleep duration and an increase in supine ahi . Correction of nsp did not affect rem duration, but supine ahi decreased after treatment . Further studies performed on a larger patient cohort may shed light on the effects of nsp and its treatment on sleep.
Sonographic estimation of fetal weight (fw), especially in late pregnancy is an important guide in obstetric care . Armed with this information informed decisions about delivery can be taken, thereby minimizing perinatal morbidity and mortality . Amniotic fluid cushions the fetus from traumatic forces, cord compression, and pathogens, as well as playing an essential role in fetal lung development . In late pregnancy, amniotic fluid production is largely dependent on fetal micturition and renal size in the newborn has been shown to bear a significant relationship to birth weight . It is therefore, reasonable to postulate a relationship between sonographically determined amniotic fluid index (afi) and estimated fw . Previous reports have investigated possible relationships between sonographically attained fluid index, and estimated fetal weight (efw) including the influence of afi on the accuracy of sonographically efw, among caucasians . However, such studies are rare among africans, especially nigerians . This study was carried out to find out if any significant relationships exist between afi and efw in a nigerian cohort of healthy pregnant women . Two hundred and fifty - eight low - risk pregnant subjects referred for routine ultrasound scans to the radiology department of the university of benin teaching hospital, nigeria were randomly selected over a 12-month period . Those with unsure dates, diabetes mellitus, and hypertensive disorders were excluded . Only those whose menstrual dates sonography was performed with a 3.5 mhz transducer (fukuda denshi; fukuda co ltd, japan). Fetal biometrics including biparietal diameter (bpd), using the cavum septum pellucidum as landmark, as depicted in figure 1; fetal trunk cross sectional area (fta) using the four chamber view of the heart, as illustrated in figure 2; and the femur length (fl), with the hook from the greater trochanter to the distal metaphysis included, as illustrated in figure 3, were measured . The fw was automatically estimated by the scanner using a combination of the bpd, fta, and fl, based on the in - built osaka university system's formula . Ultrasound scan shows how to measure fetal trunk cross sectional area, using the four chamber view of the heart . Sample report 1 shows estimated fetal weight calculated by the osaka university system's formula . Sample report 2 shows estimated fetal weight calculated by the osaka university system's formula . Data was entered into a microsoft spreadsheet and analysed using the statistical package for the social sciences (spss version 16). Measurements were stratified into pairs of afi and efw as follows; 27 - 29 weeks, 30 - 32 weeks, 33 - 35 weeks, 36 - 38 weeks, and 39 - 41 weeks . Spearman's correlation was used to test possible relationship between the afi and efw pairs . The mean age of the subjects was 29.1 4.9 years, and parity ranged from 0 to 7, with a mean of 1.5 . The mean maternal weight was 71.4 13.6 kg, and height was 1.6 0.5 m. the number of subjects in each gestational group were distributed as follows; 64 (24.8%) in the 27 - 29 weeks, 56 (21.7%) in the 30 - 32 weeks; 48 (18.6%) in the 33 - 35 weeks; 50 (19.4%) in the 36 - 38 weeks, and 40 (15.5%) in the 39 - 41 weeks; as outlined in table 1 . Amniotic fluid index and estimated fetal weight values for the gestational age ranges table 1 also shows that the mean afi and efw values for 27 - 29 weeks gestation were 172.1 mm and 1250.19 g respectively; 30 - 32 weeks were 170.3 mm and 1,648.04 g; 33 - 35 weeks were 162.3 mm and 2,273.54 g; 36 - 38 weeks were 144.0 mm and 2,906.12 g; and 39 - 41 weeks were 125.0 mm and 3,222.65 g. spearman's correlation values between afi and efw were 0.123, 0.472, 0.179, 0.210, and 0.221 for 27 - 29 weeks, 30 - 32 weeks, 33 - 35 weeks, 36 - 38 weeks, and 39 - 41 weeks respectively . There was no significant association between afi and efw for all subdivisions of gestation age, except in the 30 - 32 weeks group (p <0.05; r = 0.472). Overall, there was no statistically significant relationship between afi and efw (p> 0.05; r = 0.241). Figure 6 shows the scatter plot diagram for all pairs of afi and efw, with r value of 0.241 . Amniotic fluid disorders, oligo - hydramnios, and poly - hydramnios have been associated with intrauterine growth restriction and abnormal fetal growth, but this relationship across the entire range of fws is unclear . However, when used alone, amniotic fluid measurement has been found to perform poorly in predicting fetal distress, fetal growth restriction or low apgar scores, among others . Polyhydramnios and oligohydramnios could overestimate or underestimate sonographic fw assessment . While there are reports of afi measurements and ultrasound efws, as separate parameters, both in the nigerian and international literature,[810] there are few reports that assessed the possible relationship between afi and estimated weight, for both normal (non - diabetic) and diabetic pregnancies . This study found a decrease in mean afi values from 172.1 mm in early third trimester (27 - 29 weeks), to a value of 144.0 mm at 36 - 38 weeks, before a sharp drop to 125.0 mm at 39 - 40 weeks, as observed in previous studies . As expected increase in fw was noticed throughout pregnancy, but there was no significant association between afi and efw when all the afi and efw pairs were considered (p> 0.05; r = 0.241). Only the afi and efw pair for gestation age 30 - 32 weeks showed any significant relationship; (p <0.05). This lack of significant relationship between afi and efw across all gestational age strata is supported by the works of perni et al ., and owen et al . Possible reasons adduced for this are that swallowing and urinating mechanisms, rather than fetal size, are more involved in regulation of amniotic fluid volume . The implication of this is that fetal size may not need to be considered in variations of amniotic fluid volume across the gestational ages . It is interesting to note that kofinas and kofinas in 2012, found a significant relationship between afi and efw for both diabetic and non - diabetic pregnancies . While no explanation was offered for the former, it was postulated that fetuses of diabetic pregnancies spend more time breathing than swallowing; since swallowing and breathing are mutually exclusive, the fetuses do not swallow as much amniotic fluid as expected; thus in diabetic pregnancies, it may be necessary to consider fetal size when interpreting amniotic fluid variations across gestational ages . This present report on relationship between amniotic fluid and estimated fetal weight is probably the first among pregnant women in nigeria and therefore, raises the need for more studies on the subject, especially with larger sample sizes . It has nonetheless supported the majority of the views of similar works in the foreign literature that reported the non - dependence of amniotic fluid measurements on ultrasound estimated fetal size . This study has produced a range of values of afi and efw among nigerian africans . However, no significant relationship exists between these parameters . The probable implication of this is neither parameter merits consideration when variations in the other are considered.
Members of the genus desulfosporosinus are often found in acidic mining environments,,,,,,,,, . These bacteria may participate in metal detoxification by precipitating them in the form of sulfides . Desulfosporosinus acidiphilus and desulfosporosinus acididurans, both isolated from mining environments, represent the only two validly described, moderately acidophilic sulfate - reducing bacteria . Are also known for their tolerance to metals, in particular to copper,, . Bg was isolated from the tailings pond sediment of the bom - gorkhon molybdenum - tungsten mine in the transbaikal area . Bg to verify its phylogenetic relationship with the known acidophiles belonging to this genus and compare the putative mechanisms, which enable the bacteria to withstand acid and metal stress . Genomic dna was extracted from desulfosporosinus sp . Bg biomass using the sds - ctab method . Genomic dna was sequenced with a roche genome sequencer flx (gs flx), using the titanium xl + protocol for a shotgun library . About 189 mb of sequences with an average read length of 495 nt were generated . The reads were de novo assembled into contigs using the newbler assembler version 2.9 (454 life sciences, branford, ct). The resulting draft genome sequence of desulfosporosinus sp . Bg consists of 156 contigs longer than 500 bp, with a total length of 4,536,051 bp . Gene search and annotation were performed for all contigs longer than 500 bp using the rast server following manual curation . The draft genome of desulfosporosinus sp . Bg was around 4.52 mb, which is a relatively small size, compared to desulfosporosinus orientis 5.86 mb (nc_016584), desulfosporosinus youngiae 5.66 mb (nz_cm001441), desulfosporosinus meridei 4.87 mb (nz_cm001441.1), and desulfosporosinus acidiphilus 4.93 mb (nc_018068). However, the acidophilic desulfosporosinus acididurans has approximately the same size genome, 4.64 mb (nz_ldzy00000000.1), as strain bg . The genome includes 4516 protein - coding genes, 67 trna genes, and 9 rrna genes . The phylogenetic analysis based on concatenation of 32 ribosomal proteins showed that strain bg clustered with the acidophilic, copper - resistant desulfosporosinus sp . The closest relative of strain bg was desulfosporosinus sp . Ot isolated previously from the norilsk mining area . Several major mechanisms notable for the acid- and metal - tolerance were detected in the desulfosporosinus sp . The acid - tolerance determinants included the k - transporting atpase kdpabc (dsbg_rs17700-kdpa, dsbg_rs17705-kdpb, dsbg_rs17710-kdpc), which participates in the generation of internal positive membrane potential preventing proton influx to the cytoplasm . Strain bg along with desulfosporosinus sp . Ot has an na / h antiporter (dsbg_rs05820), known as the key transporter in maintaining the ph of actively metabolizing cells . Phylogenetic analysis shows that the antiporter was likely acquired from bacillus via lateral gene transfer . Additionally, strain bg has proton - consuming decarboxylases the arginine decarboxylase (dsbg_rs13090) and the lysine decarboxylase (dsbg_rs07860), which are involved in the mechanism of coping with low ph environment enterobacteria . It comprises an operon with transcriptional regulator csor (dsbg_rs14110) and copper chaperon copz (dsbg_rs14120). Consistent with the phylogenetic position derived from ribosomal proteins, the closest relatives of the cu - atpase of strain bg were proteins from desulfosporosinus sp . Ot (sequence similarity 97%) and desulfosporoinus sp . Two ib p - type atpases primarily responsible for translocating cd and other closely - related divalent co, hg, pb, and zn ions, were found in strain bg genome . One of the atpases (dsbg_rs14085) is present in all available desulfosporosinus genomes and located close to the cu - atpase . Interestingly, another cadmium atpase (dsbg_rs14615) has orthologous genes only in desulfosporosinus sp . I2 (88%), desulfosporosinus acidiphilus (85%), and uncultivated desulfosporosinus sp . Physiological studies did not reveal outstanding tolerance to cobalt, nickel, and cadmium in strain bg . In conclusion, we have revealed additional transporters that are not available genomes of other members of this genus . The strain has heavy metal transporters that can enable it to tolerate high concentrations of lead, mercury, or zinc . Genomic dna was extracted from desulfosporosinus sp . Bg biomass using the sds - ctab method . Genomic dna was sequenced with a roche genome sequencer flx (gs flx), using the titanium xl + protocol for a shotgun library . About 189 mb of sequences with an average read length of 495 nt were generated . The reads were de novo assembled into contigs using the newbler assembler version 2.9 (454 life sciences, branford, ct). The resulting draft genome sequence of desulfosporosinus sp . Bg consists of 156 contigs longer than 500 bp, with a total length of 4,536,051 bp . Gene search and annotation were performed for all contigs longer than 500 bp using the rast server following manual curation . The draft genome of desulfosporosinus sp . Bg was around 4.52 mb, which is a relatively small size, compared to desulfosporosinus orientis 5.86 mb (nc_016584), desulfosporosinus youngiae 5.66 mb (nz_cm001441), desulfosporosinus meridei 4.87 mb (nz_cm001441.1), and desulfosporosinus acidiphilus 4.93 mb (nc_018068). However, the acidophilic desulfosporosinus acididurans has approximately the same size genome, 4.64 mb (nz_ldzy00000000.1), as strain bg . The genome includes 4516 protein - coding genes, 67 trna genes, and 9 rrna genes . The phylogenetic analysis based on concatenation of 32 ribosomal proteins showed that strain bg clustered with the acidophilic, copper - resistant desulfosporosinus sp . The closest relative of strain bg was desulfosporosinus sp . Ot isolated previously from the norilsk mining area . Several major mechanisms notable for the acid- and metal - tolerance were detected in the desulfosporosinus sp . The acid - tolerance determinants included the k - transporting atpase kdpabc (dsbg_rs17700-kdpa, dsbg_rs17705-kdpb, dsbg_rs17710-kdpc), which participates in the generation of internal positive membrane potential preventing proton influx to the cytoplasm . Ot has an na / h antiporter (dsbg_rs05820), known as the key transporter in maintaining the ph of actively metabolizing cells . Phylogenetic analysis shows that the antiporter was likely acquired from bacillus via lateral gene transfer . Additionally, strain bg has proton - consuming decarboxylases the arginine decarboxylase (dsbg_rs13090) and the lysine decarboxylase (dsbg_rs07860), which are involved in the mechanism of coping with low ph environment enterobacteria . It comprises an operon with transcriptional regulator csor (dsbg_rs14110) and copper chaperon copz (dsbg_rs14120). Consistent with the phylogenetic position derived from ribosomal proteins, the closest relatives of the cu - atpase of strain bg were proteins from desulfosporosinus sp . Ot (sequence similarity 97%) and desulfosporoinus sp . Two ib p - type atpases primarily responsible for translocating cd and other closely - related divalent co, hg, pb, and zn ions, were found in strain bg genome . One of the atpases (dsbg_rs14085) is present in all available desulfosporosinus genomes and located close to the cu - atpase . Interestingly, another cadmium atpase (dsbg_rs14615) has orthologous genes only in desulfosporosinus sp . I2 (88%), desulfosporosinus acidiphilus (85%), and uncultivated desulfosporosinus sp . Brhc37 (90%), but not in any other available desulfosporosinus genomes . Physiological studies did not reveal outstanding tolerance to cobalt, nickel, and cadmium in strain bg . In conclusion, we have revealed additional transporters that are not available genomes of other members of this genus . The strain has heavy metal transporters that can enable it to tolerate high concentrations of lead, mercury, or zinc.
A 22-year - old - male patient presented with a painless swelling over the left eye along with mechanical ptosis of the upper lid . The mass was located anterior to the orbital rim in the superolateral part of the orbit; with the posterior edge not palpable as it was within the orbit . Anterior segment evaluation, intraocular pressure, and fundus examination of both eyes were normal . A computed tomography showed an iso - to - hypodense mass lesion in the superotemporal aspect of the left orbit, arising possibly from the lacrimal gland since the gland could not be clearly delineated from the mass . No obvious changed to the bony orbit could be noted on the scans [fig . Fine needle aspiration cytology smears drawn showed no malignant cells; however, a conclusive diagnosis could not be made . A working diagnosis of a pleomorphic adenoma of the lacrimal gland was made, and an excision biopsy was performed . Intraoperatively, the mass was pink, vascular, and well circumscribed but not encapsulated . The lacrimal gland was seen in close relation to the mass, compressed between the mass and the superolateral orbital bony rim . (a) external clinical photograph shows the mass located in the superolateral quadrant of the orbit . (b) axial slice of the computed tomography scan showing the mass in the vicinity of the lacrimal gland (yellow arrow) the cut surface was homogeneous and did not have any distended vascular channels, lacrimal tissue, or cystic dilatations . On microscopic examination, spindle cells in compact sheaves closely opposed to thin walled vessels were seen in a fibrocollagenous background [fig . The nuclei were open, oval, and contained small, uniform nucleoli [fig . . No necrosis was noted, and a few engorged vessels were seen in the outlying area . Immunohistochemical studies showed that the tumor cells stained positive for cd 34 and cd 31, which are markers for endothelium [fig . Furthermore, the tumor stained positive for smooth muscle actin and negative for cd68 [fig . Immunostaining was negative, and the ki-67 proliferation index was low with <5% of the cells staining positive [fig . Thus, the tumor had no conclusive features of a malignancy and the final diagnosis based on its microscopic appearance, and immunohistochemical characteristics was that of a solid variant of angioleiomyoma of the orbit . As it had been excised completely, no further treatment was warranted . At 1-year follow - up, no recurrence was observed . (a) low power view showing spindle cells in a background of fibrocollagenous tissue (h and e, 10). (b) high power view demonstrating uniform oval nuclei (h and e, 40) (a and b) immunohistochemical studies showed that the tumor cells stained positive for cd 34 (endothelium) and cd 31 (10) (a and b) immunohistochemical studies positivity for smooth muscle actin and immunonegativity for cd68 (10). (c) the ki-67 proliferation index was low with <5% of the cells staining positive (10) the differentials for such an orbital tumor could be cavernous hemangioma, angiomyofibroma, or complex orbital angiomyoma . The tissue of origin of angioleiomyoma is considered to be smooth muscle; so the possible native tissues from which the tumor could have arisen include blood vessels, pericytes, mller's muscle or the capsulopalpebral muscle of hessar . Angioleiomyoma is well recognized within the spectrum of vascular lesions of the soft tissues and has been subdivided into three types solid, venous, and cavernous . Since there were no dilated cavernous spaces within our tumor, we consider our case to be a solid variant . Morimoto, who classified angioleiomyomas, observed that solid variants are often painful and seen in the extremities . In contrast to his comments, our case was a solid variant which was painless . Described an angiomyofibroma of the orbit as a hybrid tumor exhibiting characteristics of a vascular leiomyoma and cavernous hemangioma . The lack of the cavernous venous channels, the unusual location, and absence of the classical purplish hue rule out cavernous hemangioma and angiofibroma in our case . Jakobiec has also described in detail the characteristics of a complex orbital angiomyoma, which had features of a lymphangiohemangioma . Three of them were located in the muscle cone, two of them were located in the superotemporal orbit, and one was located in the inferior orbit . Two other eyelid angioleiomyomas were also described in the same series . Of these eight cases, based on their histological classification, however, only one case was the same type as our case: the solid variant; five cases were of the cavernous type and two cases were venous type . Alam et al ., have also reported a similar tumor from the anterior orbit, which was found to be of the cavernous subtype . Preoperative magnetic resonance imaging may provide some clues: magnetic resonance findings of peripheral angioleiomyomas were relatively nonspecific, but t2-weighted images show a mass with mixed areas that are both hyper- and iso - intense relative to the skeletal muscle and a hypointense rim . Angioleiomyomas of the orbit are rare tumors with good prognosis, and the treatment of choice remains complete surgical excision.
Collapsing glomerulopathy (cg) was initially described in association with human immunodeficiency virus (hiv) infection and classified as a variant of focal and segmental glomerulosclerosis (fsgs). Recent studies have shown that cg is a distinct clinicopathologic entity with three variants: idiopathic, genetic and reactive . Idiopathic cg, with incidence of about 1%, has a dysregulated phenotype of podocytes with expression of proliferation marker, ki-67 . Extensive search of the available indexed english literature yielded few scattered reports of cg from our country . However, a detailed analysis of idiopathic cg in indian patients is lacking in the available literature . A study also showed a correlation of renal outcome of cg with interstitial fibrosis, high serum creatinine and heavy proteinuria . There have been no reports of a comparison of childhood and adult - onset cg with respect to the clinicopathologic features . The present study describes the clinicopathologic features of 30 patients of idiopathic cg, the largest series from this subcontinent . In addition, an attempt is made to compare these features between childhood and adult - onset cg . All native kidney biopsies diagnosed as idiopathic cg over 4 years (2006 - 2009) were retrieved . Detailed clinical and laboratory findings were reviewed . Over the duration of 4 years, 30 cases of cg were diagnosed as idiopathic among the 3314 renal biopsies evaluated in the department and were included in this study . The histologic criteria for diagnosis of cg was the demonstration of at least one glomerulus with segmental or global capillary tuft collapse, with hyperplasia and hypertrophy of the overlying visceral epithelial cells (podocytes) exhibiting features of activation . Detailed demographic and clinical data were recorded for all the cases, including clinical presentation, course of disease, therapeutic approach and renal outcome at the last follow - up . Results of laboratory investigations including viral serology (hepatitis b, c, hiv, parvovirus), tests for autoimmune diseases (antinuclear antibody, anti - dsdna, antineutrophil cytoplasmic antibody) and renal function tests (serum creatinine, urea, 24-h urinary protein excretion, urinalysis etc .) Were recorded . The renal biopsy slides (stained with h and e, periodic acid schiff, jones silver methenamine and masson's trichrome) were reviewed and the following information recorded: total number of glomeruli; proportion of globally sclerosed glomeruli, glomeruli with cg, glomeruli showing fsgs without collapse; tubular atrophy and interstitial fibrosis, interstitial inflammation, presence of tubular dilatation, casts, necrosis or degeneration; and vascular changes . The results of direct immunofluorescence examination (in all cases) and ultrastructural studies (performed in a subset of patients) were reviewed . Categorical data was analyzed using chi - square test and numerical data was compared using student's t - test . Adult - onset and childhood cases (<14 years of age at diagnosis) of cg were compared for clinical and biochemical parameters . Spss 17.0 software (spss inc ., chicago, usa) was used for performing the statistical analysis . Categorical data was analyzed using chi - square test and numerical data was compared using student's t - test . Adult - onset and childhood cases (<14 years of age at diagnosis) of cg were compared for clinical and biochemical parameters . Of the 3314 native kidney biopsies received during the study period, 30 cases were diagnosed as idiopathic cg (0.9%). Of the 30 patients, 11 were children (mean age 7 4.7 years). The duration of symptoms at the time of clinical presentation varied from 10 days to 12 months (median duration 2 months). Hypertension at presentation was noted in 18 patients (60%) while 10 patients (33.3%) had a reduction in the urinary output [table 1]. Clinical features of patients with collapsing glomerulopathy urinalysis revealed microscopic hematuria in all 30 cases (100%) with active sediments in the form of red cell casts (rbc) and/or> 30% dysmorphic rbcs in 12 cases (40%). Nephrotic - range proteinuria was noted in 16 patients (53.3%) and subnephrotic in the rest of the cases . The mean serum urea was 95.3 mg / dl (52.9 mg / dl) while serum creatinine was 4.86 mg / dl (3.93 mg / dl). The clinical and biochemical parameters were compared between childhood cg (11 cases) and adult - onset cg (19 cases). The duration of symptoms was marginally higher in pediatric patients with cg (6.5 2.1 months) compared with 4.24 2.1 months in adult patients (p = 0.053). Serum urea in children was 48.5 6.39 mg / dl compared to 115.36 13.5 mg / dl in adults (p = 0.0057) while serum creatinine was 1.88 0.25 mg / dl in pediatric patients as against 6.12 1.08 mg / dl in adult patients (p = 0.022). Quantitative 24-h urinary protein excretion was similar in both groups (3.45 1.36 g/24 h in children and 4.29 1.75 g/24 h in adults, p = 0.31) [table 2]. Clinical and biochemical parameters between childhood and adult - onset cg renal biopsy in all the included cases was adequate with a mean of 13.3 glomeruli (1.66) per biopsy . The number of obsolescent glomeruli (globally sclerosed) in these cases was 2.9 0.98 . Glomerular collapse with hyperplasia / hypertrophy of the overlying podocytes [figure 1a - d] was seen to involve a mean of 4.1 0.76 glomeruli in our study . Panel of photomicrographs showing a glomerulus with collapse (arrow) of the tuft (a) h and e, 100), better appreciated at higher magnification (b) h and e, 400 . Periodic acid schiff stain demonstrates the podocyte hypertrophy and collapse of tuft (c) pas 400 . Silver methenamine stain highlights the collapsed tuft and marked podocyte hypertrophy (d) sm 400 tubulointerstitial changes were frequent in our cases . Tubular atrophy (involving> 25% of the cortical area) was noted in 12 cases (40%) with marked atrophy (> 50% of cortical area) in two cases . Rest 18 cases showed minimal to mild tubular atrophy (<25% of cortical area). Tubular dilatation with the formation of intratubular casts [figure 2a] was seen in nine biopsies (30%). Three cases each (10% each) showed features of acute tubular necrosis and regenerative features in tubular epithelial cells . Interstitial fibrosis of variable degree was noted in all cases . The fibrosis was mild (<25% of cortical area) in 18 cases (60%), moderate (26 - 50% of cortical area) in nine (30%) and marked in three biopsies (10%). Accompanying lymphocytic interstitial inflammation photomicrograph demonstrating the tubulo - interstitial changes (a) h and e, 100 . Ultrastructural photomicrograph showing folded glomerular basement membrane with loss of foot processes of the overlying podocytes (b) uranyl acetate - lead citrate 10,000). Higher magnification better demonstrates the loss of foot processes of podocytes and microvillus transformation (c) uranyl acetate - lead citrate 16,000) the histological features were compared between childhood and adult - onset cg . Renal biopsies of pediatric patients with cg showed significantly higher proportion of glomerular segmental sclerosis without collapse (26.75 5.9% in childhood vs. 12.95 3.6 in adult - onset cg, p <0.001). Other features like proportion of glomeruli with collapse, tubulointerstitial fibrosis and interstitial inflammation were similar in both groups . These cases showed endothelial swelling, marked effacement of podocyte foot processes, degenerative changes like cytoplasmic vacuolation and microvillous transformation of podocytes . In addition, wrinkling and collapse of glomerular capillary loops [figure 2b and c] was seen in eight cases where the glomerulus involved by cg was included in the ultrathin section . The mean duration of follow - up in our cases was 12.85 months (2.3 months). Of the 30 patients, nine progressed to end - stage renal disease (esrd) requiring dialysis . These nine patients included two pediatric cases . The time to esrd from the onset of disease varied between 3 and 24 months (median 13.3 months). On statistical analysis, occurrence of esrd was not correlated with age at presentation, gender, serum creatinine and urea at diagnosis, degree of proteinuria or various histological features . Renal biopsy in all the included cases was adequate with a mean of 13.3 glomeruli (1.66) per biopsy . The number of obsolescent glomeruli (globally sclerosed) in these cases was 2.9 0.98 . Glomerular collapse with hyperplasia / hypertrophy of the overlying podocytes [figure 1a - d] was seen to involve a mean of 4.1 0.76 glomeruli in our study . Panel of photomicrographs showing a glomerulus with collapse (arrow) of the tuft (a) h and e, 100), better appreciated at higher magnification (b) h and e, 400 . Periodic acid schiff stain demonstrates the podocyte hypertrophy and collapse of tuft (c) pas 400 . Silver methenamine stain highlights the collapsed tuft and marked podocyte hypertrophy (d) sm 400 tubulointerstitial changes were frequent in our cases . Tubular atrophy (involving> 25% of the cortical area) was noted in 12 cases (40%) with marked atrophy (> 50% of cortical area) in two cases . Rest 18 cases showed minimal to mild tubular atrophy (<25% of cortical area). Tubular dilatation with the formation of intratubular casts [figure 2a] was seen in nine biopsies (30%). Three cases each (10% each) showed features of acute tubular necrosis and regenerative features in tubular epithelial cells . Interstitial fibrosis of variable degree was noted in all cases . The fibrosis was mild (<25% of cortical area) in 18 cases (60%), moderate (26 - 50% of cortical area) in nine (30%) and marked in three biopsies (10%). Accompanying lymphocytic interstitial inflammation was seen in 26 cases (86.67%). Photomicrograph demonstrating the tubulo - interstitial changes (a) h and e, 100 . Ultrastructural photomicrograph showing folded glomerular basement membrane with loss of foot processes of the overlying podocytes (b) uranyl acetate - lead citrate 10,000). Higher magnification better demonstrates the loss of foot processes of podocytes and microvillus transformation (c) uranyl acetate - lead citrate 16,000) the histological features were compared between childhood and adult - onset cg . Renal biopsies of pediatric patients with cg showed significantly higher proportion of glomerular segmental sclerosis without collapse (26.75 5.9% in childhood vs. 12.95 3.6 in adult - onset cg, p <0.001). Other features like proportion of glomeruli with collapse, tubulointerstitial fibrosis and interstitial inflammation were similar in both groups . These cases showed endothelial swelling, marked effacement of podocyte foot processes, degenerative changes like cytoplasmic vacuolation and microvillous transformation of podocytes . In addition, wrinkling and collapse of glomerular capillary loops [figure 2b and c] was seen in eight cases where the glomerulus involved by cg was included in the ultrathin section . None of the cases examined showed tubuloreticular inclusions on ultrastructural examination . The mean duration of follow - up in our cases was 12.85 months (2.3 months). Of the 30 patients, nine progressed to end - stage renal disease (esrd) requiring dialysis . The time to esrd from the onset of disease varied between 3 and 24 months (median 13.3 months). On statistical analysis, occurrence of esrd was not correlated with age at presentation, gender, serum creatinine and urea at diagnosis, degree of proteinuria or various histological features . Cg was initially described as a variant of fsgs, later shown to be associated with hiv infection and included as hiv - associated nephropathy . Later studies reported the occurrence of cg in hiv - negative patients and this entity was renamed as collapsing fsgs . However, more recent reports have suggested that cg is a distinct clinicopathologic entity, unrelated to fsgs . A recently proposed taxonomy for podocytopathies recognized three variants of cg: idiopathic, genetic or reactive . Genetic or familial cg has been described in association with arthritis or neurologic disease and mutations in coq2 have been detected in this variant of cg suggesting mitochondrial dysfunction in cg . The list of etiologies of reactive cg, which started with hiv infection, is ever - expanding with other factors including infections, medications and acute ischemia being added . Idiopathic cg is characterized by a dysregulated phenotype, manifested by loss of maturity markers such as synaptopodin, podocalyxin and re - expression of early podocyte markers (pax2 and cytokeratin) along with proliferation marker, ki-67 . The incidence of idiopathic cg in previous studies is approximately 1% . In the present study, idiopathic cg constituted 0.9% of all native kidney biopsies performed over the duration of 4 years . Review of the available indexed literature revealed a few reports of idiopathic cg from the indian subcontinent, including a report of 10 cases in adults from pakistan and another description of six pediatric cases from india . However, a detailed clinicopathologic analysis of idiopathic cg in indian patients is still lacking in the literature . This study represents the first such large clinicopathological study of 30 patients with idiopathic cg from this subcontinent . The clinical presentation of patients in our study is in consonance with the previous reports . The mean time to biopsy in our patients was 4.9 months, which is in agreement with 2.3 months in the study by laurinavicius et al . And 7.9 months reported by valeri et al . In the present study, 36.7% of the patients were children (11 out of 30) compared with 23.2% (10 of 43 patients) in the report by valeri et al . In the present study, an attempt was made to compare the clinicopathologic features of adult - onset and childhood cg . Among clinical features, childhood cg had a longer duration of symptoms prior to biopsy with lower serum urea and creatinine at diagnosis . Statistical analysis of pathologic features showed that childhood cg had a higher proportion of segmentally sclerosed glomeruli not involved by collapse (p <0.001). This is the first time that adult - onset and childhood cg has been compared with respect to clinical and pathologic features . The median renal survival of idiopathic cg is reported to be less than 18 months . In the study by valeri et al ., the patients were followed for a mean duration of 32 months during which 51% of patients progressed to esrd . In our series, the mean follow - up duration was 13 months and nine patients (30%) progressed to esrd at the end of this period . On statistical analysis, the progression to esrd was not related to age at presentation, gender, serum creatinine and urea at presentation, degree of urinary protein excretion or histological features such as percentage of glomeruli with collapse, global or segmental sclerosis ., (including hiv and non - hiv cg) showed relation of extensive interstitial fibrosis, high serum creatinine and high proteinuria with renal death . Presence of extensive collapsing lesions (> 20% of glomeruli) showed a positive effect on renal survival suggesting partial reversibility of acute lesions with appropriate / aggressive therapy . In summary, we describe the clinicopathologic features of a large series of patients with idiopathic cg . Children constituted about one - third of all patients with idiopathic cg, thus implying that timely performed renal biopsy and early recognition of this entity is imperative in this age group as well . Considering the rapidly progressive course, poor prognosis and increasing incidence in non - hiv patients, early and accurate diagnosis of cg by the renal pathologist may assist in improving the renal outcome of these patients.
The bacterial isolates were collected throughout finland during 19982007 as described (10). Of bionumerics version 5.1 software (applied maths, kortrijk, belgium) was used for sequence assembly . Allele numbers, sequence types (sts), and clonal complexes (ccs) were assigned by using the pubmlst database (5). A serum sensitivity assay was conducted with 73 c. jejuni isolates according to a described protocol (8). The same pool of serum samples from 10 healthy blood donors was used in all experiments . C. jejuni all statistical analyses were performed by using graphpad prism version 4.03 (graphpad software, san diego, ca, usa) and pasw statistics version 18 (spss inc ., chicago, il, usa). A total of 72 c. jejuni isolates from blood were successfully typed by mlst; 1 isolate had a mixed mlst pattern . Five isolates were in unassigned sts, and the rest were distributed among 11 ccs (table). Genetic relatedness of these isolates was further confirmed by using pulsed - field gel electrophoresis . * mlst, multilocus sequence typing; st, sequence type; nd, none detected . However, bacteremia episodes caused by st-677 cc isolates were exclusively diagnosed during the seasonal peak during may august (figure 1). Of c. jejuni blood culture isolates detected during may august, most (64%) were st-677 cc . Furthermore, st-677 cc was the most prevalent complex in 4 geographic regions of finland . Annual and seasonal distribution of 72 camplyobacter jejuni blood culture isolates belonging either to the st-677 clonal complex (cc) or to the other multilocus sequence typing (mlst) ccs . One isolate with a mixed multilocus sequence type was not included . C. jejuni bacteremia was diagnosed during may august (m a) or during any other month of the year (o). Susceptibility to human serum varied between c. jejuni isolates from different ccs (figure 2). St-677 cc isolates were significantly less susceptible to human serum than all other isolates (p<0.0001). St-45 cc isolates were significantly more susceptible to human serum than all other isolates (p<0.0001). Percentage of surviving bacteria in human serum for 73 blood culture isolates of campylobacter jejuni (cj), grouped according to major multilocus sequence typing clonal complexes (ccs), and for controls c. jejuni cj11168 and c. fetus . We characterized a unique collection of 73 c. jejuni isolates from blood obtained during a nationwide study in finland over a 10-year period . Despite the high population diversity of c. jejuni, nearly half of the isolates from blood showed clustering within the st-677 cc, a rare cc in other countries (12,13). Thus, invasiveness of blood culture isolates could not be solely explained by their serum resistance, although the predominant isolates of st-677 cc were more serum resistant than other isolates . C. jejuni has high st diversity . As of may 2, 2013, a total of 6,564 sts were registered (5). In this study, we detected clustering of c. jejuni isolates from blood in an uncommon st-677 cc . Further studies are needed to clarify whether bacterial characteristics might explain this finding . In our previous study, which included human fecal c. jejuni isolates obtained in finland from the mid-1990s through 2007, which is nearly the same period as in the current nationwide study, 11.7% of the isolates belonged to st-677 cc (11). The 2 most prevalent ccs in that study, st-45 cc (43.6% of fecal isolates) and st-21 cc (19.4% of fecal isolates), were detected only among 12 (16%) and 10 (14%) of blood culture isolates, respectively, in the present study . St-45 cc and st-21 cc have been shown to be prevalent in several countries (4,13). However, our results suggest that these 2 ccs are not common among c. jejuni isolates from blood in finland, which cluster more in the st-677 cc . On the basis of the present results, we speculate that st-677 cc might have a special invasive capability or has adapted to the environment in finland . In general, complement - mediated killing of serum - susceptible isolates plays a major role in restricting access of pathogens to the bloodstream . However, available information about possible serum sensitivity of c. jejuni isolates from blood is scarce (8,9). In our study of nonselected c. jejuni isolates from blood, susceptibility to human serum varied according to mlst cc . In conclusion, in this nationwide study during a 10-year period in finland, we found by mlst analysis that half of the bacteremia isolates of c. jejuni clustered within an otherwise uncommon st-677 cc . Whether this finding indicates special adaptation of st-677 cc to finland or to the human bloodstream our findings emphasize the role of using well - defined clinical materials in studies on bacterial pathogenicity and severity of human disease.
A retrospective western blot study was performed for all confirmed bse cases diagnosed in france after july 1, 2001, as previously described (13). Briefly, prpres was extracted using the tesee western blot confirmatory assay (bio - rad, marnes - la - coquette, france) according to the manufacturer s instructions . For immunoblotting, antibodies rb1, 6h4 (r - biopharm, st . Didier au mont dor, france), sha31 from the tesee western blot (bio - rad), and saf84 (kindly provided by j. grassi, commissariat energie atomique, saclay, france) were used; these antibodies recognize the bovine prp sequences 110113, 156164, 156163, and 175180, respectively . Two western blot assays were conducted on the brainstem samples and used either an antibody against the prp core (rb1, 6h4, or sha31) or a c - terminal antibody (saf84) (13). The characteristics of h - type and l - type bse (6,13) that were sought included 1) a higher or lower molecular mass of unglycosylated prpres with core antibodies in h - type and l - type bse compared with c - type bse, 2) a lower proportion of diglycosylated prpres in l - type bse, and 3) presence of an additional band at 14 kda with saf84 in h - type bse . All cases with features of h - type or l - type bse were then subjected to further western blot analyses for detailed quantitative analysis of prpres molecular features (apparent molecular masses and glycoforms proportions). Among the 645 bse cases confirmed between july 1, 2001, and july 1, 2007, 7 h - type and 6 l - type isolates were identified; these had occurred at a frequency of 03 h - type and 1 l - type case per year, compared with 6219 cases of c - type bse per year (table). Molecular typing of 48 of these 645 samples was not possible because low levels of prpres prevented detailed molecular characterization or because sample amount was insufficient . All 13 atypical cases were detected in cattle> 8 years of age, from fallen stock (9 cases) or abattoir (4 cases); not 1 atypical case was found among the 98 bse cases detected by clinical surveillance during this period . These 13 atypical cases were diagnosed by the different rapid bse tests routinely used in france: 9 by prionics - check western, lia, or priostrip (aes, combourg, france), 3 by elisa bio - rad, and 1 by idexx herdchek (idexx laboratories, schiphol - rijk, the netherlands). During retrospective interviews, the farmer and veterinarian for 6 of these animals reported clinical signs consistent with tse in 3 fallen stock . This series of 13 cases identified since the beginning of exhaustive active surveillance should be compared with a total of 17.1 million adult cattle tested, of which 3.6 million were> 8 years of age . In addition to these 13 cases, the first case of atypical (h - type) bse was identified in 2000, during an exhaustive active surveillance program in rendering plants in a limited region of france . * h - type, higher molecular masses of unglycosylated protease - resistant prion protein (prpres); l - type, lower molecular masses of unglycosylated prpres; c - type, classic bse; uc, unclassified . The distribution of bse - infected cattle by birth date (figure 1) shows that 1 or 2 (h- or l - type cases) were eventually found positive by rapid tests of the brainstem in each annual birth cohort from 1986 through 1997, which compares with up to 221 cattle infected with c - type bse born during 19902001 for which bse was diagnosed during 20012007 . All h - type and l - type bse cases showed similar features (figure 2) regarding the high apparent molecular masses of unglycosylated prpres (mean difference of 1 kda using 6h4 antibody) for h - type bse and lower levels of diglycosylated prpres (mean difference of 35% using 6h4 antibody) for l - type bse, compared with c - type bse . For l - type bse cases, the differences in apparent molecular masses were more obvious for the diglycosylated band (1-kda difference using 6h4, compared with 0.3 kda for the unglycosylated band), as previously observed (6). The 14-kda band characteristic of h - type bse was similarly detected in the 8 isolates with saf84 antibody but in none of the other cases classified as l - type or c - type bse . Distribution of bovine spongiform encephalopathy cases identified from july 1, 2001, through july 1, 2007, by year of cattle birth . H - type, higher molecular masses of unglycosylated protease - resistant prion protein (prpres); l - type, lower molecular masses of unglycosylated prpres; c - type, classic bse . Representative western blot analyses of protease - resistant prion protein (prpres) in h - type (lanes 2, 3), l - type (lanes 5, 6), and c - type (lanes 1, 4, 7) cases of bovine spongiform encephalopathy (bse). Bars to the left of the panels indicate the 29.0-, 20.1-, and 14.3-kda marker positions . H - type, higher molecular masses of unglycosylated prpres; l - type, lower molecular masses of unglycosylated prpres; c - type, classic bse . Monoclonal antibodies sha31 and saf84 were used for prpres detection in panels a and b, respectively . This study involved exhaustive molecular typing of bse cases during a given test period and in a country in which bse testing has been mandatory for all adult slaughtered or fallen cattle . France tests more animals than any other european country; 30% of the animals tested in the european union are tested in france . The estimated frequency of h - type and l - type bse was 0.41 and 0.35 per million adult cattle tested, respectively (1.9 and 1.7 in cattle> 8 years of age). Given the implementation dates of measures to control bse and the birth dates of these bse - infected cattle, foodborne exposure to an infectious agent cannot be fully excluded for any of these cattle . However, the distribution of cattle affected by h- and l - type bse, by year of birth, differs strikingly from that of cattle affected by c - type bse and is consistent with the hypothesis that h - type and l - type bse might represent sporadic prion disorders . In comparison with another sporadic prion disease, the annual frequency of sporadic creutzfeldt - jakob disease in humans, which is estimated only by analyses of reported clinically suspect cases, is 12 cases per million . Similar studies in other countries, instead of those free of c - type bse, would be useful . An alternative hypothesis to foodborne contamination, such as contamination by a scrapie agent, cannot be fully excluded, as such contamination has been shown to be a risk factor for scrapie in sheep (14). This study relied on the identification of bse cases by examination of the brainstem only, as derived from our knowledge of c - type bse (15). We cannot exclude possible differences in the pathogenesis of atypical bses that might result in underestimation of their frequency, e.g., involvement of the brainstem at a later stage than with c - type bse . This possibility is at least suggested by data available for l - type bse, which shows a preferential distribution of abnormal prp in more rostral brain regions (4,12). Studies of the pathogenesis of these novel bse forms are thus important for understanding of prion disorders of domestic ruminant species.
Hallucinations that occur in more than one modality simultaneously and emanate from a single source are called multimodal hallucinations (mmhs). We report mmh in two patients of treatment - resistant schizophrenia and its successful management with clozapine . A 50-year - old male with a diagnosis of treatment - resistant schizophrenia had a continuous illness for 30 years . His psychopathology was characterized by delusions of control, made acts, somatic passivity, and hallucinations in different modalities . The patient used to hear voices in isolation and could hear the voices of multiple unknown males and females coming from a distance of few meters, discussing and commenting on him in a derogatory manner . While hearing the voices, he also visualized the unknown male and female figures in the external space in the background of his surroundings . These figures were seen as vividly as other real objects, making gestures, lip movements, and uttering the words . He would have these experiences in clear consciousness and was experiencing them every day for few hours since many years . Many times, he had acted out on these hallucinations, leading to significant dysfunction . Similarly, but less frequently, the patient also reported experiencing the smell and taste of certain food items simultaneously in clear consciousness in the absence of any external source . Would describe the taste and smell to be pleasant at times and unpleasant at other times . In addition to these experiences, the patient also had hallucinatory experiences in different modalities occurring from different sources at different times but not simultaneously . Full blood picture, peripheral smear, liver and renal functions, blood glucose levels were within reference limits, and a computed tomography scan of the head was normal . In view of treatment resistance, he was started on clozapine and the dose was gradually increased to 300 mg / day over 4 weeks with regular monitoring of adverse effects . Brief psychiatric rating scale (bprs) score before starting clozapine was 63 and at the end of 3 months of treatment, it reduced to 37 (hallucinatory behavior score decreased from 7 [extremely severe] to 3 [mild]). It has now been 9 months since starting clozapine, and the patient is sustaining improvement . A 23-year - old male presented with a continuous illness of 8 years suggestive of treatment - resistant schizophrenia . He could see a doll - like figure of a human being (culturally accepted entity) in clear consciousness in addition to his immediate surroundings . He could visualize the doll - like figure as vividly as other real objects and could see the movements of its different body parts as well . At the same, he would hear commanding verbal auditory hallucinations originating from that source with synchronized lip movements . These verbal auditory hallucinations would threaten him of adverse consequences if he did not follow the instructions . He had these experiences every day for 6 years and had acted out on these voices . He had attempted suicide thrice and homicide once over last 6 years under the influence of these hallucinations . He was started on clozapine, and the dose was gradually increased to 300 mg / day over 4 weeks with regular monitoring of adverse effects . Bprs score before starting clozapine was 70 and at the end of 3 months of treatment, it reduced to 28 (hallucinatory behavior score decreased from 7 [extremely severe] to 1 [not present]). A 50-year - old male with a diagnosis of treatment - resistant schizophrenia had a continuous illness for 30 years . His psychopathology was characterized by delusions of control, made acts, somatic passivity, and hallucinations in different modalities . The patient used to hear voices in isolation and could hear the voices of multiple unknown males and females coming from a distance of few meters, discussing and commenting on him in a derogatory manner . While hearing the voices, he also visualized the unknown male and female figures in the external space in the background of his surroundings . These figures were seen as vividly as other real objects, making gestures, lip movements, and uttering the words . He would have these experiences in clear consciousness and was experiencing them every day for few hours since many years . Many times, he had acted out on these hallucinations, leading to significant dysfunction . Similarly, but less frequently, the patient also reported experiencing the smell and taste of certain food items simultaneously in clear consciousness in the absence of any external source . Would describe the taste and smell to be pleasant at times and unpleasant at other times . In addition to these experiences, the patient also had hallucinatory experiences in different modalities occurring from different sources at different times but not simultaneously . Full blood picture, peripheral smear, liver and renal functions, blood glucose levels were within reference limits, and a computed tomography scan of the head was normal . In view of treatment resistance, he was started on clozapine and the dose was gradually increased to 300 mg / day over 4 weeks with regular monitoring of adverse effects . Brief psychiatric rating scale (bprs) score before starting clozapine was 63 and at the end of 3 months of treatment, it reduced to 37 (hallucinatory behavior score decreased from 7 [extremely severe] to 3 [mild]). It has now been 9 months since starting clozapine, and the patient is sustaining improvement . A 23-year - old male presented with a continuous illness of 8 years suggestive of treatment - resistant schizophrenia . He could see a doll - like figure of a human being (culturally accepted entity) in clear consciousness in addition to his immediate surroundings . He could visualize the doll - like figure as vividly as other real objects and could see the movements of its different body parts as well . At the same, he would hear commanding verbal auditory hallucinations originating from that source with synchronized lip movements . These verbal auditory hallucinations would threaten him of adverse consequences if he did not follow the instructions . He had these experiences every day for 6 years and had acted out on these voices . He had attempted suicide thrice and homicide once over last 6 years under the influence of these hallucinations . He was started on clozapine, and the dose was gradually increased to 300 mg / day over 4 weeks with regular monitoring of adverse effects . Bprs score before starting clozapine was 70 and at the end of 3 months of treatment, it reduced to 28 (hallucinatory behavior score decreased from 7 [extremely severe] to 1 [not present]). Various terms such as mmhs, multiple hallucinations, polysensory hallucinations, and dissociative hallucinations have been used to describe hallucinations occurring in more than one modality . These terms have described different permutations and combinations of source and timing of the hallucinations occurring in more than one modality . Reviewing the heterogeneity in the phenomenon described by these terms, chesterman and boast operationally defined mmh as mentioned above . The hallucinatory experiences occurring in more than one modality but at different times (e.g, visual hallucinations in the morning and auditory hallucinations in the evening) are considered sequential instances of unimodal hallucinations . The occurrence of simultaneous hallucinations as the major manifestations of a psychiatric disorder have been often dismissed as factitious disorder or malingering . In addition, many terms used to describe simultaneous hallucinations such as dissociative hallucinations carried the disadvantage of implying a particular diagnosis . Actually, mmhs have been reported in many organic conditions and severe mental disorders including schizophrenia . About 50% of the patients with schizophrenia were found to have simultaneous auditory and visual hallucinations . In addition, few published reports of mmh occurring in other psychotic disorders are also available . Both of the above patients had mmh involving auditory and visual hallucinations . In addition, one of them had mmh involving smell and taste, and to our knowledge appears to be the first of its kind . Another important finding in this report is that both of our patients with mmh were treatment resistant, and responded well to clozapine . We could not find any data on the association between the presence of mmh and treatment resistance in schizophrenia . Whether the presence of mmh suggests greater severity of disease process and higher chances of treatment resistance needs to be evaluated . Both of our patients responded to clozapine and the study on the differential efficacy of antipsychotics in schizophrenia subjects with mmh may shed more light on the treatment of such subjects . The available data on neurobiology of mmh in psychotic disorders is derived from few case reports and a study with small sample size (n-10). These reports have consistently shown that cortical activation during mmh follows the sensory hierarchy, with consistent activation of heteromodal cortical regions involving temporoparietal junction, posterior superior temporal sulcus, and inferior parietal region . Jardri et al . Found superior temporal sulcus activation to be specific for mmh . In these studies, subjects primarily had mmh involving visual and auditory modalities . It would be interesting to find the cortical activation during mmh involving other sensory modalities in different permutations and combinations . The neurobiology and effect on course, treatment resistance, and prognosis of schizophrenia need elucidation by systematic research with adequate sample size.
The potential links of coffee consumption to various health conditions including cancer have been studied extensively . While most early studies were concerned that coffee consumption was carcinogenic and would lead to higher rates of cancers, subsequent efforts to control for other factors and comprehensive review studies found that although many case - control studies linked coffee consumption to increased rates of cancer, multiple larger prospective trials found no connection [14]. After many additional studies, it was concluded in 2008 that there is a convincing level of evidence that there is no relationship between coffee intake and breast cancer, and a probable level of evidence that there is no relationship between coffee intake and pancreatic cancer, ovarian cancer, thyroid cancer, prostate cancer, bladder cancer, bone health, cardiovascular disease, and reproductive adversities . However, for a number of other cancers, including colorectal cancer, the evidence of a relationship with coffee is rated possible, suggesting further research is warranted . Most of the accumulated evidence for colorectal cancer indicates that higher daily coffee consumption is associated with reduced incidence of colorectal cancer [57]. The most recent meta - analysis of the association between coffee consumption and colorectal cancer conducted separate analyses for case - control evidence and cohort study evidence and found that when comparing the highest category of coffee consumption to the lowest category, high coffee consumption was associated with a significantly reduced risk of colorectal cancer (or = 0.85, 95% ci 0.75, 0.97). When combining 16 cohort studies, high coffee consumption trended towards a slight reduction in the risk of colorectal cancer (or = 0.94, 95% ci 0.88, 1.01). Contrary to the above findings, which found the strongest overall relationship between coffee and colon cancer, a 2010 study that only focused on prospective cohort studies found no relationship between coffee consumption and risk of colon cancer . Since the meta - analyses described above, data from three large prospective cohort studies have explored this question, with one study finding a reduced risk of colorectal cancer for those with high coffee intake, one study finding no significant differences, and the third finding coffee to be associated with an increased risk . Thus, although most studies and meta - analyses have concluded there is some evidence of a negative relationship between coffee consumption and colorectal cancer, conflicting evidence from a well - designed meta - analysis and recent large cohort studies highlights the need for additional research in this area . All of the recent studies and meta - analyses state that the relationship remains unclear [510], and evidence suggesting a stronger protective effect among women should be explored . Our current objective is to examine the relationship of daily consumption of caffeinated and decaffeinated coffee to colorectal cancer in a large observational cohort of older women in the united states (us). Hypothesized mechanisms such as phenolic phytochemicals cannot be explored in this study, but caffeinated versus decaffeinated coffee will be studied, along with coffee preparation methods that may impact the amount of fiber reaching the gastrointestinal tract . The women's health initiative is a large, ongoing national health study of women in the us aged 5079 at inception . The women's health initiative consisted of three overlapping clinical trials and an observational study . Since the original study period, two extensions have allowed for ongoing collection of follow - up data from consenting participants . The current study uses data from the women's health initiative observational study (whi os) and the follow - up extensions . The whi os design and measures have been described in detail in previous publications [12, 13]. In summary, 93,676 women between the ages of 5079 were recruited from october 1993 through december 1998 at 40 clinical recruitment centers regionally distributed across the us . To be eligible, participants also had to be postmenopausal and be unlikely to die or move in the next 3 years . From the original 93,676, we excluded 959 participants who had a history of colorectal cancer at baseline and an additional 773 participants with missing coffee data . Finally, we removed 8,166 participants with missing model covariate data and/or no follow - up time, giving a final analysis sample of 83,778 . Among this sample demographic data, lifestyle, and medical history were collected using self - report questionnaires at a baseline visit for all enrolled participants . Physical measurements such as height and weight were collected by trained staff at a baseline clinical visit . Coffee intake was measured at baseline by a question asking how many cups of regular coffee (not decaf) do you usually drink each day? (count tall [12 ounces . Or more] cups and espresso drinks made with double shots of espresso as 2 cups .) Response categories were none; 1 cup; 2 - 3 cups; 4 - 5 cups; 6 or more cups . Decaffeinated coffee was measured the same way, replacing the word regular with decaffeinated . Tea consumption was measured with the same type of question but referred only to caffeinated tea . Any indication of possible colorectal cancer was verified by a review of medical records by trained physician adjudicators at each site before being confirmed at the whi clinical coordinating center via blinded review . More details on the identification of historical and incidental colorectal cancer cases are provided in previous publications . Information on various types of bowel screening was collected by self - report questionnaire during annual assessments . Baseline characteristics of the sample by coffee intake (table 1) are presented with means with standard deviations for continuous variables and frequencies with percentages for categorical variables . Statistical comparisons between groups are done with t (continuous variables) and chi - square (categorical variables) tests for differences by colorectal cancer . Differences in linear trends of coffee drinkers by demographic characteristics were assessed by fitting either a linear (continuous / ordinal variables) or logistic (dichotomous variables) model with the demographic variable as a function of linear coffee (0 cups / day = 1;> 0<4 cups / day = 2; 4 cups / day = 3). Proportional hazards modeling was used to evaluate the relationship between coffee variables and colorectal cancer (table 2), following participants from enrollment in the whi observational study until they either had an event, died, or were lost to follow - up . Prior to modeling, the proportional hazards assumption was checked by fitting models with each colorectal cancer outcome as a function of coffee intake and its interaction with the log survival time . Modeling was done in two ways, first with each outcome as a function of regular coffee intake unadjusted, then adjusted for age, ethnicity, education, alcohol use, smoking / pack - years, bmi, physical activity, total energy intake, red meat intake (oz . ), fruit / vegetable intake (cups), percent caloric intake from fat, fiber intake (g), calcium intake (diet plus supplements, mg), hormone use (with type if current), nsaid use, history of treated diabetes, and family history of colorectal cancer . Separate models were then used to create accompanying linear trend p values, substituting the categorical coffee variable for a linear version (0 cups / day = 1;> 0<4 cups / day = 2; 4 cups / day = 3) with p values for the linear coffee term presented . To examine whether results may differ for decaffeinated and total coffee consumption, we ran sensitivity analyses substituting each for regular caffeinated coffee in our primary models (appendix tables a and b in supplementary material available online at http://dx.doi.org/10.1155/2016/6918431). Caffeinated tea consumption was also tested as a possible covariate . To examine effects of adjustment variables on colorectal cancer, full model results of the three - level regular coffee variable are also included (appendix table c). To get a graphical look at colorectal cancer survival by coffee groups, in addition to the primary models, additional models were run to determine whether the effect of coffee on colorectal cancer varied by subgroup variables of age, ethnicity, alcohol use, smoking / pack - years, ht use, ht type (among those using ht), bmi, and coffee type . For each subgroup a proportional hazards model was fit modeling colorectal cancer as a function of coffee intake, the subgroup of interest, their interaction, and all adjustment variables used in the primary models . . Characteristics of our sample are presented by colorectal cancer status table 1 . During the mean follow - up of 12.9 years, 1,282 (1.53%) participants had documented cases of colorectal cancer . Older age, higher bmi, less physical activity, higher fat intake, not using hormone replacement therapy, a history of treated diabetes, and a family history of colorectal cancer were all significantly associated with incident colorectal cancer . Characteristics of our sample are also presented by level of coffee consumption in table 2 . Participants with higher coffee consumption tended to be slightly younger, were more likely to be white, and were less likely to have a college degree . They also were more likely to be a current smoker and/or alcohol drinker and have a higher bmi . Prior to modeling we checked the proportional hazards assumption and found no significant interactions between the primary coffee variable and the log survival time for any of our outcomes, indicating that the proportional hazards assumption has not been violated in our models . In unadjusted models, we explored whether the relationship of coffee consumption with colorectal cancer differed when including or removing decaffeinated coffee from the consumption variable . Thus, the consumption variable in the final models included only regular (caffeinated) coffee . Very few study participants were regular tea drinkers and this variable was not significantly related to cancer incidence or coffee consumption . Three - level regular coffee consumption was associated with colorectal cancer, with results for both low and high coffee intake relative to non - coffee drinkers (hr 1.18, 95% ci 1.051.33; hr 1.22, 1.001.47, resp .) (see table 2). After multivariate adjustment, this association remained significant (p = 0.04) but the confidence interval for 4 + cups per day was of borderline nonsignificance . In the multivariate model for 3-level coffee consumption (see appendix table c), significant predictors of colorectal cancer included age (hr 1.32, 95% ci 1.261.37 for a 5-year increment), education (hr 1.26, 1.081.47; and hr 1.23, 1.051.44 for higher education groups versus high school / ged), smoking (hr 1.34, 1.151.56 for past, 20 pack - years versus never smokers; hr 1.63, 1.262.11 for current, 20 pack - years versus never smokers), bmi (hr 1.10, 1.051.15 for a 5 kg / m increase), current hormone use (hr 0.72, 0.620.84 for e - alone versus never, and hr 0.75, 0.640.89 for e + p versus never), current nsaid use (hr 0.87, 0.770.97), and a positive family history of colorectal cancer (hr 1.24, 1.081.43). We also examined the relationship between coffee consumption and the three main types of colorectal cancer (table 2). Like all colorectal cancers together, colon cancer occurred more often among those who drank 4 + cups of coffee per day . The interaction effects of coffee consumption levels by covariate groups (age, ethnicity, alcohol use, smoking, current ht use, current ht type, bmi, and coffee brew type) on colorectal cancer were examined and only coffee brew type was borderline nonsignificant (p = 0.06). Participants who primarily consumed drip coffee had relatively stable hazard ratios relative to non - coffee drinkers regardless of the amount of consumption . Non - drip coffee drinkers, however, had increased hazard ratio for colorectal cancer relative to non - coffee drinkers as coffee consumption increased . The results of our unadjusted model suggest that drinking regular caffeinated coffee, both in moderate amounts (13 cups / day) or in larger amounts (4 + cups / day) was associated with an increased risk of colorectal cancer in postmenopausal women . After controlling for covariates in the multivariate model, only the group reporting coffee consumption between 0 and 4 cups per day remained statistically significant, while the group reporting more than four cups per day was of borderline significance, despite the large sample size . This finding contrasts with other literatures on the association of coffee consumption with colorectal cancer, which suggests a protective effect of coffee, especially in larger amounts such as 4 - 5 or more cups daily [57]. The most recent meta - analysis for the association between coffee consumption and colorectal cancer conducted separate analyses for case - control evidence and cohort study evidence and distinguished between colorectal cancer, colon cancer, and rectal cancer . When combining the results of 25 case - control studies, they found that when comparing the highest category of coffee consumption to the lowest category, high coffee consumption was associated with a significantly reduced risk of colorectal cancer (odds ratio (or) = 0.85, 95% ci 0.75, 0.97) and colon cancer (or = 0.79, 95% ci 0.67, 0.95) but not rectal cancer alone (or = 0.95, 95% ci 0.79, 1.15). When combining 16 cohort studies, high coffee consumption trended towards a slight reduction in the risk of colorectal cancer (or = 0.94, 95% ci 0.88, 1.01) and colon cancer (or = 0.93, 95% ci 0.86, 1.01) but not rectal cancer alone (or = 0.98, 95% ci 0.88, 1.09). Subgroup analyses in the case - control studies indicated that the relationship between coffee consumption and reduced risk of colorectal cancer was significant among women and europeans while the relationship between coffee consumption and reduced risk of colon cancer was significant among europeans . However, in the cohort studies, the relationship between coffee consumption and reduced risk of colon cancer was significant only for asian women . Contrary to the above findings, which found the strongest overall relationship between coffee and colon cancer, a 2010 study that only focused on prospective cohort studies found no relationship between coffee consumption and risk of colon cancer . Since the meta - analyses described above, data from a large prospective study of older adults with a mean follow - up of 10.5 years showed that when compared to coffee nondrinkers, there was a reduced risk of colon cancer among people who drank 4 - 5 cups of coffee daily (hr = 0.85, 95% ci: 0.75, 0.96) and those who drank 6 or more cups daily (hr = 0.74, 95% ci: 0.61, 0.89). When broken down by caffeinated and noncaffeinated coffee, results for drinkers of caffeinated coffee were similar to the overall results, while the effect was smaller and only trended towards significance for drinkers of decaffeinated coffee . In 2013, a large prospective cohort study of 57,398 men and women in the us did not find any significant associations between coffee intake and risk of colorectal cancers, despite exploring many subgroups and variables that have been significant elsewhere . Finally, in 2014, a large prospective cohort study of 58,221 japanese men and women found an increased risk of colon cancer for men drinking 2 - 3 cups per day (hazard ratio (hr) = 1.26) and men drinking 4 or more cups per day (hr 1.79). No effect was found for women, but very few japanese women in this study were coffee drinkers . In summary, although the majority of studies have found a protective effect of higher coffee intake with respect to colorectal cancer, a number of recent high quality studies have found no effect [8, 9, 16], while others found an increased risk of cancer as was found in our current study of older women . Our study is different from most others in that it focuses only on women . When breaking all colorectal cancers into the subtypes of colon, rectal, and rectosigmoid, we did not find any significant relationship between regular coffee consumption . With a total of 1,282 participants with colorectal cancer identified, dividing those into cancer subgroups and then examining by 5 groups of coffee consumption by brew method resulted in subgroups with small sample sizes, limiting statistical power . Despite this limitation, there was some indication or trend towards a higher incidence of colon cancer in participants drinking more than four cups of coffee and using nondrip brew methods . Like our primary findings, this contradicts recent meta - analytic results which suggest a possible protective effect of higher coffee consumption for incident colon cancer . Multiple mechanisms by which coffee (both caffeinated and decaffeinated) may impact colorectal cancer have been postulated and studied, but few firm conclusions have emerged . Although many research studies focus mainly on caffeine content, coffee contains over a thousand different chemical compounds including phenols, melanoidins, and diterpenes that have antioxidant effects and may have health benefits . One recent study showed that caffeic acid and not caffeine suppressed cancer cell growth in colon cancer . Other research concludes that protective effects of coffee against colorectal cancers are likely due to the high concentrations of polyphenols and dietary fiber that reach the colon . One of the unique contributions of this study is that we were able to include information on the method by which coffee was brewed in our analyses . Very few other analyses include this information, and our results show that the brewing method for coffee was related to incidence of colorectal cancers . Specifically, drinkers of more than four cups of coffee per day who did not use drip coffee methods were more likely to get colorectal cancer . This contradicts the potential mechanisms proposed by some researchers, who posited that because nondrip filters allow more dietary fiber and potentially antioxidants to be released into the coffee, colon motility may be increased, potentially resulting in reduced risk of colorectal cancers . Since europeans use the drip method and paper filters less often than in the united states, the finding of reduced risk among europeans was seen as possible support for this theory . Our data suggest that it is important for future research to study the method of coffee preparation when examining the relationship between coffee consumption and colorectal cancers . The most recent research in this area has identified over 20 biomarkers associated with coffee consumption in the prostate, lung, colorectal, and ovarian (plco) cancer screening trial study . Some of these biomarkers have been linked to reduced rates of colorectal cancer, and are thus promising targets for research on the mechanisms by which coffee might affect colorectal cancers . Another recently published study of colorectal cancer recurrence found that japanese men drinking 3 or more cups of coffee daily had reduced recurrence of proximal colorectal cancer, yet coffee consumption was also associated with increased incidence of distal colorectal cancer recurrence . A strength of our study is that any indication of cancer was followed up by a review of medical records by study physicians and adjudicators . Although the study was not designed to produce a nationally representative sample, whi had 40 clinical research centers recruiting in 24 states across the us and was able to enroll large numbers of women from groups that typically are harder to recruit, minority women and women aged 7079 . While no other studies focused exclusively on women, the incidence of cancer in our study (0.118% annually) seemed quite comparable to the incidence (0.136% annually) in another large prospective study that also included men . Conversely, a possible limitation is that the participant pool is not fully representative of all women but was limited to postmenopausal women, the age at which colorectal cancer is most prevalent . The questions grouped coffee consumption into 5 categories (none; 1 cup; 2 - 3 cups; 4 - 5 cups; 6 or more cups) instead of using a continuous scale . The questionnaire did not ask about herbal tea usage, which is also frequently studied in published analyses similar to ours . It is also possible that there are other confounding factors that were not measured or omitted from analyses . One such possible factor, aspirin use, has been linked to incidence of colorectal cancer in the past, but a prior study with the whi cohort found no relationship . Data from the whi os show that coffee consumption is related to a slightly higher incidence of colorectal cancer . Existing evidence has been inconclusive, and the current results add further to the conflicting data . Data showing that using a nondrip brew method for preparing coffee may elevate the risk of developing colorectal cancers appears to be a new finding that contradicts theories put forward on other manuscripts and should be explored further.
Globally, the disciplines of medicine and dentistry use indices of health widely and these have been developed for many different purposes . Uses include the classification of conditions to aid the understanding of aetiology, risk, prognosis and treatment outcome (sharabiani et al ., 2012). They can also be used to determine prevalence and or incidences within a population, and therefore, help in the planning and provision of treatment at the individual or population levels . In recent years, their use in the planning of services, particularly within cash - limited, publicly funded health services such as the uk national health service, has gained greater acceptance . Our own experience within orthodontics is that indices have been used to prioritize treatment to those most in need and likely to benefit from orthodontic treatment, as well as to monitor the quality of treatment outcome . In the prioritization of treatment provision, we have become familiar with the use of the index of orthodontic treatment need (iotn) (brook and shaw, 1989), which has been in routine use in nhs primary care in england and wales since 2006, and somewhat earlier than this in many secondary care settings (holmes and wilmott, 1996). This index was developed with the aim of prioritizing the functional need for treatment through its dental health component (dhc) and psychosocial need through the aesthetic component (ac). This was not the first such index used for this purpose in orthodontics, with indices such the handicapping labio - lingual deviations index (draker, i960), the treatment priority index (grainger, 1967), the handicapping malocclusion assessment record (salzmann, 1968) and the occlusal index (summers, 1971) all having been developed earlier than the iotn . The iotn itself is a modification of an index previously developed by the swedish dental health board (linder aronson, 1974). In the process of developing an index of health the principal factor is its intended purpose, but ease of use in daily practice is also important, since it may involve the collection and interpretation of a large amount of data from which, a single useful indicator is then provided (arvaniti and panagiotakos, 2008). In addition, an index should be both valid and reliable . A number of studies have been carried out to assess the validity and reliability of the iotn, as well as the time taken to use the index . Validity is often measured against expert opinion . In comparison with other orthodontic occlusal indices, the strengths of the iotn dhc component are, not only its validity and reliability, but also that it is quick and easy to use (cardoso et al ., 2011). Moreover, the grading also appears to be unaffected by age, at least within the relatively narrow age range of the adolescent patient (cooper et al ., 2000). As a result, it is not only widely used in orthodontic research (bellot - arcs et al ., 2012), but is also highly rated by those involved in planning orthodontic service provision within the uk (de oliveira, 2003). The iotn dhc is a straightforward five - point scale, with the greatest need for treatment classified as being group 5 and little or no need for treatment classified as group 1 . Within each group, there are well defined descriptors of the features of the malocclusion deemed as indicators of orthodontic need (such as overjet, impacted teeth and missing teeth). The reason the index is quick and easy to apply is that the malocclusion is scored simply on the worst feature . In order to identify this feature in a systematic manner it is suggested the assessor uses the acronym mocdo (missing teeth, overjet, crossbites, displacement of contact points, overbite) (richmond et al ., 1994). Currently within the nhs, orthodontic treatment is limited to iotn dhc groups 4 and 5, and group 3 where the ac is grade 6 or above . Although widely used, there are some limitations of the iotn . In the case of the ac of the index, it comprises only class i and class ii division 1 incisor relationships and there are no class ii division 2 or class iii incisor relationships . In the case of the dhc, some of the functional indications for orthognathic treatment are not included, or might be classified differently if the malocclusion were not treatable with orthodontics alone . For example, excessive upper labial segment show at rest is not included in the iotn in the absence of any other occlusal traits, and yet this can lead to potential gingival and periodontal problems and might only be amenable to treatment with combined orthodontic and orthognathic treatment . With these limitations in mind, it was decided to create a new index of orthognathic functional treatment need (ioftn), using wherever possible the same traits as used in the iotn dhc, but with modifications and additions to reflect the functional indications of treatment need for orthognathic patients . In this way, it was hoped to create an index that feels familiar to those using the iotn, is valid, reliable and quick and easy to use . Using the iotn dhc as a starting point, four consultant orthodontists with extensive experience in treating orthognathic patients (aji, sjc, mtc, nph) devised a draft ioftn based on a five - point scale ranging from very great need for treatment (5) through to no need for treatment (1). The draft index was then presented at the british orthodontic society consultant orthodontists group (cog) symposium in bristol during march 2013, following which it was formally circulated to all 280 members of the cog for written comment . Forty - six members replied with written comments and these were then reconsidered at a further meeting of the panel of experts . Modifications were made to the wording of the index to reflect the comments and at this point the index was considered to have face validity . The panel of four experts then worked in pairs to score 163 sets of start study models using the new index . The sample of models represented various malocclusions that had previously been treated using an orthognathic approach . The scores were then compared and, wherever there was disagreement, the panel discussed them and came to a consensus score for each of the 163 sets of models . At this point, the index (figure 1) was considered to have content validity, as it was felt that all of the possible facets of the construct of whether or not an orthognathic treatment approach was appropriate for functional reasons, had been considered . The index of orthognathic functional treatment need (ioftn) twenty - one specialist orthodontists with experience of orthognathic treatment, all consultant orthodontists and senior fttas were then asked to score the 163 sets of models using the new index, in order to test agreement with the expert panel scores . The scores were then analysed using cohen s kappa for inter - operator agreement with the expert panel scores when assigning the patient to one of the five major categories . It also looked at assignment within the major categories to the individual sub - categories (for example, having decided a patient was in category 5, what was the agreement for allocation to the subcategories within the major category). In addition, the percentage agreement with all of the categories scored by the experts was also determined for each of the 21 assessors . In order to determine the intra - operator agreement, two consultants scored 50 sets of study models on two separate occasions 1 week apart . The results were analysed using spss (ibm spss statistics 220; ibm corp ., chicago, il, usa) and stata version 13 (stata corp ., college station, tx, usa). Table 1 is the summary table of the 163 study models, illustrating the number of models in each of the ioftn categories . The kappa scores for inter - operator agreement with the expert panel scores for the major categories are illustrated in table 2, and this shows good to very good agreement for all raters . The percentage agreement of the 21 assessors with the expert panel scores for all categories is illustrated in figure 2 and ranged from 681 to 92% . The per cent agreement was over 80 for 16 of the 21 assessors, which can also be considered good . When the agreement for the subcategories within the major categories was compared with the expert panel score, it was also good to very good for all raters (table 3), 25 . There were too few models in category 1 for statistical testing as few patients in this category will have undergone orthognathic treatment . Histogram illustrating the percentage agreement of each of the 21 assessors with the expert panel scores for all of the 23 categories within the ioftn the weighted kappa scores for intra - operator agreement were 053 for operator 1 and 080 for operator 2, showing moderate to good agreement over time for each rater . In recent years, there has been a drive to reduce costs within the uk nhs, not only to reduce overall spending, but also to divert money and resources from what are deemed low priority treatments, to those deemed to be of higher value and where the evidence to support their use is said to be greater . As far back as 2006, primary care trusts in england responsible for nhs funding within their areas began compiling lists of what they considered low priority treatments . One such list, the croydon list has received much attention and comprised 34 treatments . Other pcts compiled much longer lists of over 100 procedures and this prompted the audit commission in 2011 to suggest that their implementation in commissioning health could lead to annual savings to the nhs of 500 million (audit commission, 2011). Although the audit commission found some commonality in the lists, there was not complete uniformity . What could be considered low priority in one area in 2012, the south central pcts, in consultation with solutions for public health, investigated the evidence to support the routine funding of orthognathic treatment for reasons of function, sleep apnoea, speech and temporomandibular joint dysfunction . Following this investigation, the southern cluster within the south centrals area decided that all orthognathic treatment should be considered to be of low priority, except for severe sleep apnoea, cleft lip and palate and following major trauma (hiow / ship priorities committee april 2008 to march 2012). The northern cluster also considered it to be low priority and decided not to fund treatment for speech or temporomandibular joint dysfunction, but were prepared to continue funding for functional reasons and sleep apnoea, and provided the patients were categorized as iotn 4 or 5 (solutions for public health, 2012). It was at about the same time that the strategic health authorities in england were abolished, in line with the introduction of the uk government s health and social care reforms (ham, 2012), and the south central pcts commissioning intents appeared to have been lost during this nhs restructuring . In the new era, the commissioning of all dental services and for the interim, all oral and maxillofacial services, were now to be commissioned centrally by nhs england and implemented locally by the local area teams . In late 2013, nhs england published its interim clinical commissioning policy for orthognathic treatment . Although this interim policy was withdrawn in march 2013, it had stated the following (british association of oral and maxillofacial surgeons (baoms), 2014): the iotn must be 4 or 5; functional symptoms must have an important impact on quality of life, which would normally have become apparent within 5 years of achieving skeletal maturity; the multidisciplinary team confirms that orthodontic treatment is insufficient by itself to adequately correct these functional symptoms; patients have reached skeletal maturity; orthognathic treatment should be low priority on the grounds of insufficient evidence of functional improvement for: jaw pain, particularly that associated with the temporomandibular joint . It would seem that the interim guidance was based on the earlier south centrals pct work and included the iotn as a measure of severity and functional need . However, the use of iotn has limitations as a measure of functional and health need in orthognathic treatment provision . In particular, some severe dentofacial deformities and malocclusions would not be eligible for nhs funding for orthognathic treatment using iotn . Examples include excessive upper labial segment gingival exposure with evidence of gingival and/or periodontal effects, complete scissor bites or facial asymmetries with marked effects on the occlusal plane . It is in order to overcome these limitations with the use of iotn in orthognathic treatment provision that the ioftn was developed . The new ioftn has good face and content validity and also demonstrates good to very good inter - operator agreement (064088), similar to the iotn (07310797) (brook and shaw, 1989). This is perhaps not surprising, in that the two indices share a similar format, which clinicians are familiar with . As with the iotn it is important, particularly when scoring from study models alone, that additional information is provided; for example, information would be required regarding the degree of upper labial segment exposure where present, or functional effects such as trauma to the soft tissues where there is an increased overbite . This will not be a problem where the ioftn is used to score the patient at the chairside . Similar limitations also apply to the use of iotn when scoring more routine malocclusions from study models alone . Once again, the ioftn also demonstrates moderate to good intra - operator agreement over time (053080), not too dissimilar to that observed with the iotn, with its reported kappa scores of 075084 (brook and shaw 1989). After 24 years of service to orthodontics, it is perhaps timely that the application of iotn in clinical practice is being revisited . The concept that any one index should not be expected to fit all eventualities when deciding on treatment priorities has recently been made in reference to secondary care orthodontics (cousley, 2013). We therefore feel that the ioftn is a natural evolution of the iotn that should be used when setting treatment priorities for combined orthodontic and orthognathic care . It is both valid and reliable and, like the iotn, is quick and easy to use, thereby fulfilling the essential requirements of an index . However, the ioftn concerns the functional indicators for orthognathic treatment, and other clinical and psychological indicators will also be important in the assessment of orthognathic patients . A new index, the ioftn, has been developed to help in the prioritization of severe malocclusions not amenable to orthodontic treatment alone . The index has face and content validity and has been shown to have good inter and moderate to good intra - operator reliability . As a result of being an evolution of the iotn, the format is similar to this index and so it should be easy to incorporate within daily orthognathic practice . Contributors each of the authors contributed in the following manner: substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; drafting the article or revising it critically for important intellectual content; and final approval of the version to be published.
This results in rapidly progressive and abnormal structural changes in the eye that can causes blindness from retinal detachment, macular folds, refractive amblyopia, or strabismic amblyopia.1 in 1987, a joint statement by the american academy of pediatrics, american academy of ophthalmology, and american association for pediatric ophthalmology and strabismus was released recommending rop screening by an experienced ophthalmologist for all infants with a birth weight <1500 g or with gestational age <32 weeks, as well as for select infants with a birth weight between 1500 and 2000 g believed to be at high risk by the attending neonatologist.2 despite advances in detection and treatment, rop continues to be a leading cause of childhood blindness and visual impairment worldwide in extremely premature infants with low birth weight.234 retinopathy of prematurity is a leading cause of childhood blindness in the middle east . Advances in neonatal care, improved access to healthcare, and improved delivery of healthcare especially related to maternal and child healthcare has resulted in better survival of preterm children . However, these improvements have resulted in an increase in the rate of rop and severity at presentation.5 screening premature infants for rop at salmaniya medical complex (smc) in bahrain began as a program in 1996 for all premature infants with a birth weight 1600 and a gestational age 32 weeks . The present study reports the incidence of rop from january 2002 to january 2011 among all premature infants admitted to the infant care unit at smc . This medical complex is the main government secondary and tertiary hospital in bahrain to asses the applicability of the current rop screening criteria and to compare our data with international data . This retrospective study, including all infants with a birth weight 1500 and a gestational age 32 weeks who were admitted to the neonatal intensive care unit (nicu) at smc and were examined 46 weeks after birth from 2002 to 2011 using the american association for pediatric ophthalmology and strabismus guidelines (1997).2 this was performed by reviewing the screening forms attached to the medical record [figure 1]. Data were collected via chart review on gestational age, birth weight, clinical information, rop stage, rop zone, and the presence of other disease . Fundus examination details and staging of rop screening form a pediatric ophthalmologist (1, 3) performed all examinations . Data were collected by searching the nicu database, which included clinical information about neonates and rop . Infants were included in this study if they were born at smc or any of the ministry of health maternity hospitals and transferred and admitted to the nicu at smc, and were examined and monitored by a pediatric ophthalmologist . The international classification of rop and american academy of pediatrics recommendations were used for classification of the findings.6 the findings were classified according to the maximum stage of rop in either eye . Examinations were performed at the main neonatology unit in bahrain, which has four consultant neonatologists, a capacity of 50 neonates, and a nicu capacity of seven cases . The neonatology unit strictly implements oxygen management and monitoring with a target of 85% to 92% oxygen saturation . Oxygen saturations are maintained at stable lower levels and avoiding fluctuations for very - low - birth - weight infants . If rop was not noted, eye examinations were performed until vascularization reached zone 3 of the retina . It was the responsibility of the medical and nursing staff to ensure that the examinations were scheduled at the appropriate ages based on the guidelines . To examine the peripheral retina, amethocaine 1%, cyclopentolate 0.5%, or tropicamide 0.5% was instilled with phenylephrine 2.5%, and a sterile eyelid specula and scleral depressor were used as necessary [figure 2]. Preparations of the premature baby for fundus examination examination of premature baby with indirect ophthalmoscope the pediatric ophthalmologist documented the vascularity of the retina and the stage of rop on the rop screening form, and indicated the timing of the follow - up . Incidence of rop in the study population was determined and analyzed on the basis of birth weight.7 for analysis, follow - up examinations for those with rop were screened at intervals indicated by the severity of the disease . Infants were monitored until a diagnosis of rop was established as rop or another disease . The cryo - rop trial presented a threshold of treatment for rop which was defined as at least five clock hours of contiguous, or eight cumulative clock hours of stage 3 rop in zone i or ii in the presence of plus disease.8 infants were classified according to the birth weight into group 1 who were <1000 g, and group ii, who were between 1000 and 1500 g. the data were collected using microsoft excel software (microsoft corp ., redmond, wa, usa). The data were transferred to statistical package for social studies (spss 22) (ibm, chicago, usa) for analysis . Univariate analysis was performed using a parametric method for calculating frequencies and percentage proportions of qualitative variables . For continuous variables mean and standard deviations were calculated . To compare rop risk in subgroups, the study population included 1829 premature infants of which 1795 (98.6%) infants were included in the study because their data in the patient charts were complete . There were 700 [38.9%]) infants in group 1 and 1095 [61.1%]) infants in group ii . Stage iii rop was detected in 68/1795 (3.8%) infants and represents 18.5% of the total number of rop cases . Stage iii threshold disease requiring laser retinal photocoagulation was detected in 21/367 (5.7%) infants, and 5/367 (1.4%) infants progressed to stage iv rop which required vitreoretinal surgery . Eighty percent (293/367) of the rop cases occurred in neonates with bw <1000 g [table 1]. Stages of rop by birth weight the average estimated gestational age (ega) at birth was 25 weeks (range, 18 weeks to 32 weeks). The mean birth weight was 1047 g (range, 5941500 g) [table 2]. Stages of rop by birth weight and gestational age the ratio of premature infants 1500 g per live births (159,501) was 1.2% from 2002 to 2011 . The total number of premature cases 1500 admitted to the neonatal unit at smc represents a mean of 92.6% of all premature infants born in bahrain during the study period from 2002 to 2011 . The overall incidence of any rop among all newborn infants in bahrain during the study period was 1/434 (0.2%) infants . Mean gestational age and birth weight were significantly lower among infants with rop [table 3].9 distribution of live births per year in the national registry and premature infants in bahrain and at the smc our study represents the incidence of rop in the largest study population in bahrain to date . The incidence of rop in previous studies varies by region from 22% to 56% [table 4].10111213141516 the incidence of rop in the current study is similar to previous reports . However, comparison to other studies is tenuous at best as some studies included neonates with a birth weight <1250 g. this could result underestimating the incidence of rop compared to our study . In addition, other studies have included pre - threshold criteria based on the early treatment for retinopathy of prematurity (etrop) study,16 which treated some patients sooner than the defined threshold for cryo - rop with good results . The most important change is that treatment can now be considered sooner without regard to the number of clock hours involved . The new criteria defined by etrop and the clinical algorithm shows that in most circumstances,17 peripheral retinal ablation should be considered for any eye with type i rop, zone i rop with plus disease (regardless of stage), zone i rop with stage 3 disease (with or without plus disease), zone ii rop, stage 2 or 3, with plus disease . Data comparison between different studies gilbert et al.18 compared gestational age and birth weight between countries with low, moderate, and high levels of development . In highly developed countries, they found that all infants with severe rop had a mean gestational age <26 weeks and a mean birth weight <800 g. mean gestational age and birth weight of infants with severe rop in the present study were 25 weeks and 730 g, respectively . Birth weight and gestational age in the rop group were significantly lower than in the non - rop group . These findings are consistent with other studies that birth weight and gestational age are important factors associated with rop . In some recent studies, extremely premature infants with lower gestational age had a higher incidence of type 1 rop . No infant with a gestational age> 26 weeks at birth or birth weight> 1000 g had type 1 rop.19 birth weight may not influence the incidence of type 1 rop in extremely premature infants.20ahin et al . Was found no association between type 1 rop and bw in extremely preterm infants with a ga of <28 weeks.21 rop is a potentially avoidable cause of blindness in children . The proportion of blindness resulting from rop varies greatly among countries and is influenced both by levels of neonatal care (in terms of availability, access, and neonatal outcomes) and the availability of effective screening . Gilbert et al.22 suggest that the population of infants who develop severe rop in highly developed countries differs from those who are affected in less well - developed countries, given the complex interaction between case mix, neonatal care, and survival rates, as well as variation in screening practices and follow - up rates of discharged infants, and retreatment programs . First, the study was conducted among in patients in the nicu and thus could misrepresent the general incidence of rop . Second, premature infants were referred to neonatal units at other ministry of health hospitals once they were stabilized systemically, and although most of the infants returned to our hospital for follow - up, we were unable to access complete documentation . Advances in electronic documentation and follow - up that were recently introduced at the hospital will provide more clinical information and a better infrastructure for prospective studies to investigate the incidence of rop in relation to the risk factors . Rop cases were associated with birth weight <1000 g. the incidence of rop in bahrain in 20022011 correlates with other published data.
Gastrointestinal stromal tumours (gists) are rare tumours of the gi tract, making up 0.21% of gastrointestinal malignancies . First identified by mazur and clark in 1983, these mesenchymal tumours can occur anywhere along the gastrointestinal tract [1, 3]. Relative rarity combined with non - specific presentation results in delayed diagnosis [4, 5]. We present the first case of gist leading to intussusception at the jejuno - ileal junction in an otherwise well patient prior to presentation . A 47-year - old female presented as an emergency with severe abdominal pain and profuse vomiting . She had been experiencing symptoms of intermittent vague abdominal pain associated with weight loss over 6 months, but no change in bowel habit . Pyrexial and abdominal examination revealed distended abdomen with localized tenderness in the left iliac fossa . Blood tests revealed an elevated white cell count (18.8), c - reactive protein (25), microcytic anaemia (hb 108) and a mildly raised lactate (2.37). She was commenced on intravenous fluids . An abdominal plain film showed prominent bowel loops and ultrasound of the abdomen revealed numerous loops of a distended, fluid filled, non - peristalsing bowel . There was some evidence of bowel wall thickening, but no clear source of obstruction was identified . A ct scan was performed showing mesenteric vascular gas and a target sign, virtually pathognomonic of intussuception [5, 6] (figs 1 and 2). The patient underwent a laparotomy, which revealed small bowel obstruction with evidence of impending perforation . A pedunculated polypoid lesion at the jejuno - ileal junction was identified as the lead point for intussusception . The intussuscepted segment of small bowel was resected and end - to - end primary anastomosis formed . Histological findings were in keeping with a 75-mm gist, jejuno - ileal in origin, with a mitotic count of <5/mm and a moderate (24%) risk of recurrence . The tumour was positive for cd117 and carried a c - kit mutation with a deletion in exon 11 . Intussusception is the telescoping of a proximal loop of bowel and its mesentery (intussusceptum) into the lumen of bowel distal to it (intussuscipiens). The phenomenon is rare in adults and presents with non - specific symptoms, such as recurrent abdominal pain, nausea and vomiting . Up to 90% of adult cases have a defined lesion acting as the lead point, which is malignant in overhalf of cases [1, 6]. While correct diagnosis is based on operative findings, suspicion may be raised following imaging techniques . Ct with both oral and intravenous contrast is now widely recognized as the most accurate diagnostic tool (58100% accuracy in a recent case series) [1, 4]. Alternating hyper- and hypodense layers of the bowel wall of the intussuscepted segment give rise to a classical gists are rare tumours demonstrating a broad spectrum of invasiveness [5, 7] and unpredictable behaviour, with recent suggestion that these tumours should no longer be classified as benign or otherwise . Classically gists grow exophytically, into the peritoneal cavity or adjacent organs, further decreasing their likelihood of causing an intussusception . Rarely, however, gists can grow as a pedunculated polyp, as in our case, which may go on to act as a lead point for an intussusception . Their growth pattern leads to a range of non - specific symptoms (much like an intussusception), meaning gists often remain unnoticed until advanced enough to cause complications such as ulceration, obstruction or gi bleed . Gists originate from a malignant transformation of the interstitial cells of cajal [1, 3]. 95% of cases show a mutation in the kit proto - oncogene, setting them apart from leiomyomas, leiomyosarcomas and schwannomas of the small intestine . Furthermore, near universal expression of cd117a product of the kit proto - oncogene which makes up part of the kit transmembrane receptor tyrosine kinase is found . Again following the relatively recent discovery of this immunohistochemical profile, management of gists has been transformed . Imatinib, a targeted tyrosine kinase inhibitor, has revolutionized treatment since its introduction in 2001 . Universal guidelines for its use are not currently available; however, it has a clear role in both adjuvant therapy and neoadjuvant therapy for unresectable or borderline resectable tumours [1, 3, 9, 10]. Prior to the introduction of imatinib, surgery was the only treatment available and to this day offers the only chance of cure . Surgery is offered for all tumours over 2 cm in diameter, along with a role in debulking or symptomatic relief . Below 2 cm the natural history of these rare tumours is relatively unknown; therefore, treatment planning is difficult . Given the rarity of spread to lymph nodes, regional lymphadenectomy has not been found to be useful . Intussusception is a rare finding in adults, even more so when occurring secondary to a gastrointestinal stromal tumour . We present the first case of a jejuno - ileal intussusception secondary to a gist in an otherwise well patient . Diagnosis can be difficult given the non - specific nature of the symptoms, and while different imaging modalities may be useful in evaluating these patients, a firm diagnosis cannot be made until histological analysis . Curative treatment can only be offered by surgery; however, imatinib has a role in both adjuvant and neoadjuvant treatment.
The shoulder joint is a joint that relies on interactions and balance among multiple joints1, 2 . The shoulder joint relies more on muscles than on bones or ligaments to support the shoulder and maintain the stability of the shoulder3 . Stabilization relates to the human ability to consciously or subconsciously control large or small movements occurring in the joints4 . The movements of the shoulder bone and the humeral bone are controlled by the coupling force of two muscles: the rotator - cuff muscle and the elevator muscle of the scapular . If this coupling force collapses, the muscle - coordination activity does not occur and the rotator cuff is compressed5 . In this case, the humeral head only partially maintains contact with the articular fovea, leading to many pathologic conditions, such as shoulder instability3, 6 . The activity of the scapular stabilizer muscles is important in counteracting the instability that occurs due to the aforementioned structural problems . The scapular stabilizer muscles include the serratus anterior, the upper trapezius, and the lower trapezius7 . In particular, the activity of the serratus anterior muscle is very important for scapular movements and control8, 9 . Extensive electromyography (emg) studies of the stabilizer muscles that contribute to shoulder stabilization have been carried out . The muscle activity of the serratus anterior and that of the upper trapezius have been shown to be imbalanced in subacromial - impingement syndrome, subacromial bursitis, rotator - cuff laceration, and rotator - cuff and biceps - brachii tendinitis10 . Closed - chain exercises are frequently used for shoulder - joint dysfunction prevention and in rehabilitation programs11 . Closed - chain exercises are frequently used in therapeutic exercise programs to generate dynamic stability in the joints and to maintain posture, since these exercises not only improve strength and endurance, but also induce the co - contraction of many muscles through mechanical compression of articular surfaces eliciting larger proprioceptive responses by stimulating the afferent receptors around the joints12 . Along with closed - chain exercises, sling exercises, which require performing exercises while hanging on swinging ropes, are one of the treatment methods that can be used to prevent injuries, since these exercises stimulate the proprioceptive senses to strengthen weakened muscles and give the necessary stimuli to maintain joint stability . Kirkesola13 reported that if vibrations of the rope were given during sling exercises, instability would be increased, further stimulating the proprioceptive sensory organs in the joints, and incorrect neuromuscular control could be corrected, reducing muscle imbalance . Therefore, the present study compared the muscle activity of the upper trapezius with those of the serratus anterior, and the lower trapezius when slings, unstable surfaces, were laterally vibrated, to examine the effects of vibration during sling exercises on the shoulder stabilization muscles . The subjects of the present study were 15 adult men without any history of damage to the shoulder girdle or shoulder pain . The subjects average age was 26.13 5.13 years, their average height was 174.00 5.44 cm, and their average weight was 73.47 13.46 kg . All subjects read and signed a written consent form . The subjects performed push - up exercises with knee flexion . Al.1 advised that push - up exercises with knee flexion produce emgs similar to those produced during push - up exercises with knee extension, while eliciting less force than push - up exercises with knee extension . Before emgs were measured, the subjects performed push - up exercises with their hands placed on the floor at shoulder width, their knees placed on the floor, and their elbow joints extended . The subject performed push - up exercises on an unstable surface (sling) and maintained isometric contraction for five seconds in a state of scapular protraction and elbow extension, in the final stage of the motion, and emg signals from the serratus anterior, the upper trapezius muscle, and the lower trapezius muscle were collected during state of isometric contraction . In the second exercise, the subject performed push - up exercises on a sling and maintained isometric contraction in the last stage, and the rope of the sling was manually vibrated during the isometric contraction . Vibrations was given within a range of 10 cm for five seconds at a frequency of 1 hz, in time with a metronome . In the third and fourth exercises, vibrations of 3 hz and 3.5 hz, were respectively applied . Usa) was used to measure muscle activities during the exercises . For the measurement, electrodes were attached to the upper trapezius, the lower trapezius, and the serratus anterior . 18.0), and one - way anova was used to compare muscle activities among the various vibration frequencies on the unstable surface . The upper trapezius and the lower trapezius did not show any significant differences among isometric contraction without vibration, isometric contraction with vibration of 1 hz, and isometric contraction with vibration of 3 hz, but showed significant differences at isometric contraction with vibrations of 3.5 hz . The serratus anterior showed a significant differences between isometric contraction with vibration of 3 hz and isometric contraction with vibration of 3.5 hz (table 1table 1 . Comparison of muscle activation among the various vibration frequencies (unit:% rvc)ic1 hz3 hz3.5 hzut0.034 0.0130.034 0.0130.035 0.0130.057 0.010lt0.025 0.0080.027 0.0070.027 0.0070.053 0.009sa0.051 0.0120.050 0.0110.083 0.0140.083 0.014p<0.05, mean sd, ic: isometric contraction, ut: upper trapezius, lt: lower trapezius, sa: serratus anterior). * p<0.05, mean sd, ic: isometric contraction, ut: upper trapezius, lt: lower trapezius, sa: serratus anterior the coupling force of the upper trapezius, the serratus anterior, and the lower trapezius is the core of scapular movements14 . If this muscle recruitment is changed, or the motions of these muscles are limited by an antagonist, the movement pattern will collapse . It has been revealed that in shoulder impingement syndrome, patients shoulders are elevated, the muscle activity of the upper trapezius increases, and the muscle activity of the serratus anterior decreases . In particular, the serratus anterior, which is an important muscle scapular movement imbalance, has been studied by many researchers . Fatigue of the serratus anterior decreases scapular rotation and protraction, and displaces the humeral head in a forward and upward direction1, 10, 11 . Therefore, it may cause secondary impingement syndrome and rotator - cuff tear, decrease the range of scapular motion, or disturb the contraction timing of the serratus anterior, thereby inducing stress in the glenohumeral joint15 . If abnormal scapular movements appear, due to problems in the serratus anterior, the position of the fossa would be affected, and the force and alignment involved in movements around the glenohumeral joint would eventually be affected16 . Kirkesola14 reported that when the rope used for sling exercises was vibrated, instability increased further stimulating the proprioceptive sensory organs in the joints, and incorrect neuromuscular control could be corrected, reducing muscle imbalance . As the load - bearing surface becomes more unstable, the muscle activities of the serratus anterior and the lower trapezius would increase, selectively strengthening the scapular - stabilizer muscles, reducing muscle imbalance . Exercises on a sling elicit significantly higher muscle activities than exercises on a fixed surface . Therefore, exercises on a sling should simultaneously strengthen many muscles that contribute to shoulder joint stability, increasing the stability of the shoulder complex . In the present study, to examine the effects of diverse vibration frequencies on the upper trapezius, the lower trapezius, and the serratus anterior during exercises on a sling, an unstable surface, vibration frequencies of 1 hz, 3 hz, and 3.5 hz were used . In the present study, all three muscles showed gradual activity increases as the frequency of vibration increased from 1 hz to 3 hz and 3.5 hz . The upper trapezius and the lower trapezius showed significant changes in muscle activity at 3.5 hz, and the serratus anterior showed prominent changes in muscle activity at 3 hz and 3.5 hz . Therefore, as the vibration frequency increased, making the bearing surface more unstable, the recruitment of the upper trapezius, the lower trapezius, and the serratus anterior increased . To perform exercises that will selectively strengthen the serratus anterior,
However, there is substantial variation in gastric cancer incidence with the highest rates reported from korea, japan and eastern europe, whereas western europe and the us have low incidence rates . The global burden of gastric cancer is shifting rapidly from the developed world to the developing world . Recent evidence indicates that a large number of genetic and epigenetic alterations in oncogenes, tumor suppressor genes and genetic instability as well as telomerase activation determine a multi - step process of gastric carcinogenesis [1, 2]. However, these molecular events found in gastric cancer differ depending upon the two histological types, intestinal and diffuse types of gastric carcinoma, indicating that intestinal and diffuse type carcinomas have distinct genetic pathway . The evolution of intestinal tumors is generally characterized as progressing through a number of sequential steps including gastritis, intestinal metaplasia, dysplasia and carcinoma, while no preceding steps are detected in the pathogenesis of diffuse carcinoma other than the obvious chronic gastritis . The most significant advance in the pathogenesis of gastric cancer is involved with the recognition of the role of h. pylori as the most important etiological factor for gastric cancer . Both the complex host / microbial interaction and growth factor / cytokine networks induced by h. pylori are mediated by the microenvironment that is an indispensable player in the multistage process of gastric carcinogenesis . This review provides an overview of h. pylori - induced inflammation and the molecular and cellular events of tumor microenvironment that underlie gastric cancer . There are several of h. pylori - virulence associated genes such as caga, vaca, icea and baba . Among them, the caga gene is localized at one end of the cag pathogenicity island (pai). The cagpai encodes components of a type iv secretion system, by which the caga gene product, caga, is delivered into gastric epithelial cells . The caga - positive h. pylori strains are associated with higher grade of gastric inflammation and higher risk of gastric cancer than the caga negative strains . Reported that caga+/vacas1 + strains of h. pylori that are blood group antigen - binding adhesion (baba2) positive are associated with activity and chronicity of gastritis . Adherence of h. pylori via baba2 may play a key role for efficient delivery of vaca and caga . Moreover, hatakeyama recently reported that caga binds an src homology 2 (sh2) containing tyrosine phosphatase shp-2 in a tyrosine phosphorylation - dependent manner and activates the phosphatase activity of shp-2 . Deregulation of shp2 by caga is an important mechanism by which caga positive h.pylori promotes gastric carcinogenesis . Moreover, hatakeyama found that east - asian caga shows stronger shp2 binding and greater biological activity than western caga . Differences in the biological activity of western and east - asian caga proteins, which are determined by variation in the typrosine phosphorylation sites, may underlie the different incidences of gastric cancer in these two geographic areas . In addition to deregulation of shp2 by caga, h. pylori is a potent activating factor of nf - kb in gastric epithelial cells . Activation of nf - kb by h. pylori infection induces a variety of gene expression including cytokines (il-1, il-6 . Il-8, tnf alpha), vegf, cox2, inos, cell - cycle regular, mmp2, mmp9 and adhesion molecules [1012]. Successful eradication of h. pylori leads to down regulation of cox2 in the epithelial and stromal cells . High expression of cox2 mrna, protein, and enzymatic activity is detected in the tumor cells of gastric cancer . Importantly, cox2 activity is induced by a variety of mediators including inflammatory cytokines such as tnf alpha, interferon - gamma and il-1 . Cox2 facilitates tumor growth by inhibiting apoptosis, maintaining cell proliferation and stimulating angiogenesis within cancer cells . H. pylori infection produces reactive oxygen and nitrogen species that cause dna damage, followed by chronic gastritis and intestinal metaplasia . Nitric oxide generated by inos is converted to reactive nitrogen species that bring about direct dna mutation such as p53 and protein damage, inhibition of apoptosis, and promotion of angiogenesis . Goto et al . Reported that the expression of inos and nitro tyrosine in the gastric mucosa was significantly high in h. pylori infected patients who developed gastric cancer at least two years after the initial biopsies . Three major factors, including environmental factors (diet, obesity, cigarette smoking), host factors (h. pylori strains), and genetic factors, cooperatively affect the genesis of gastric cancer . Of these factors, host genetic factors play a critical role in susceptibility to gastric carcinogenesis . El - omar reported that pro - inflammatory il-1 gene cluster polymorphisms increase the risk of gastric cancer and its precursors in the presence of h. pyroli . Moreover, the pro - inflammatory il-1 genotypes are associated with an increased risk of both intestinal and diffuse types of gastric cancer . In addition, tnf alpha and il-10 genotypes are viewed as independent risk factors for gastric cancer . The combination of three or four pro - inflammatory cytokine polymorphisms affecting il-1 beta (il-1b-511 t), il-1 receptor antagonist (il-1rn*2), tnf alpha (tnf - a-308a) and il-10 (il-10 haplotype ata) results in a highly significant increased risk of development of gastric cancer . However, we recently found that il-10 haplotype (il-10 ggcg) is associated with increased risk of gastric cancer and high levels of plasma il-10 serum in atomic bomb survivors . More recently, ohyauchi et al . Reported that il-8 polymorphism is also associated with increased risk of gastric cancer in the japanese population . These host genetic factors as well as h. pylori strains may determine why some individual infected with h.pylori develop gastric cancer while others do not . H. pylori infection induces stem cell hyperplasia in chronic inflammation leading to increased mutation and dna methylation . Stem cells in the gastric epithelium have yet to be precisely identified, but there are several proposed markers of normal human stem cells in the gastrointestinal tact including musashi1 (msi-1), hes-1 and nuclear beta - catenin . Msi-1 is an rna binding protein that may be necessary for stem cell maintenance and/or asymmetric cell division . In human gastric epithelium, msi-1 is expressed at the proliferative regions of the antrum and fundic glands but its expression is decreased in intestinal metaplasia suggesting that msi-1 is a marker of gastric progenitor cells . Interestingly, h. pylori infection strongly induces the expression of msi-1 that is correlated with h. pylori density . Ushijima group recently reported that h. pylori infection potently induces methylation of cpg islands (lox, hand1, thbd and p41arc), suggesting that h. pylori induces dna methylation in stem cells . The rna component of telomerase, hterc, and the catalytic subunit, htert, are essential and the minimum components that are required for telomerase activity . In fact, we found that low levels of telomerase activity exist in ki-67-positive epithelial stem cells, which simultaneously express htert protein and hterc protein in intestinal metaplasia of the stomach . The levels of htert expression and telomerase activity are in parallel with degree of h. pylori infection, suggesting that telomerase competence of stem cell hyperplasia caused by h. pylori infection may be linked to chronic mitogenesis that can facilitate increased mutagenesis . The approach for cancer therapy targeting telomerase and/or telomeres has the potential for innovative anti - cancer drugs . Telomestatin, a g - quadruplex stabilizing agent found in natural products, is highly selective for telomere g - quadruplex structures formed by the 3 overhang compared to other compounds . Telomestatin preferentially induces apoptosis in cancer cells or not in normal fibroblasts and epithelial cells . Moreover, kondo et al . Recently reported that loss of heterozygosity and histone hypoacetylation of the pinx1 gene, a possible telomerase inhibitor, are associated with reduced expression in gastric carcinoma . They also revealed that telomerase activity is inhibited with trichostatin (tsa) and nicotinamide (nam) in gastric cancer cells even if htert expression is not changed . Currently, a body of evidence indicates that bone marrow - derived stem cells can engraft and differentiate into nonhematopoietic cells of ectodermal, mesodermal and endodermal tissues other than hematopoietic tissues . Recently, taketo group found that smad4-deficient mouse colorectal cancer cells produce cc chemokine ligand 9 (ccl9) and recruit the mmp - expressing immature myeloid cells (imc) that express cc chemokine receptor 1 (ccr1) and that lack of ccr1 prevents the accumulation of mmp - expressing imc at the invasion front and suppresses tumor invasion . These exciting results suggest that human gastric cancers also may have the possibility to exhibit interaction between cancer cells and bone marrow - derived stromal cells implicated in tumor invasion, as majority of human gastric cancer is associated with loss or dysfunction of tgf - beta signaling . It should be examined whether or not bone marrow - derived cells in gastric cancer microenvironment produce mmp and then encourage tumor cells to invade into the stroma . Moreover, gastric cancer originating from bone marrow - derived cells has been reported in mouse models . However, cogle et al . Reported that bone marrow stem cells mimic cancer but do not initiate it in human cancers . Multiple genetic and epigenetic alterations in oncogenes, tumor suppressor genes, cell cycle regulators, cell adhesion molecules and dna repair genes, as well as genetic instability and telomerase activation are responsible for tumorigenesis and progression of gastric cancer [1, 2, 31]. Hmhl1, cdh1, rar - beta2, ps2 and runx3 by dna methylation is involved in two distinct major genetic pathways of gastric cancer [2, 3, 34]. Hypermethylation of the p16 and of hmlh1 promoters is preferentially associated with intestinal type gastric carcinoma, whereas concordant hypermethylation of the cdh1 and rar - beta2 promoters is predominantly detected in diffuse type gastric carcinoma . Loss or reduction of runx3 and ps2 expression by promoter methylation is a common event in both types of gastric carcinoma . In addition to promoter methylation, acetylated histone h4 is reduced in the majority of both types of gastric carcinoma . Histone h4 is progressively deacetylated from the early stage to the late stage of the multi - step carcinogenesis of the stomach . Moreover, histone h3 acetylation is associated with reduced p21 (waf1/cip1) expression by gastric carcinoma . Among these epigenetic events, runx3, a runt domain transcription factor involved in tgf - beta signaling, is silenced in 63% gastric cancer as well as in 73% of hepatocellular carcinoma, 70% of bile duct cancer, 75% of pancreas cancer, 62% of laryngeal cancer, 46% of lung cancer, 25% of breast cancer and 23% of prostate cancer and 5% of colon cancer . Interestingly, inactivation of runx 3 by promoter methylation is found in 8% of chronic gastritis, 28% of intestinal metaplasia, and 27% of gastric adenoma, but not in chronic hepatitis b . These findings suggest that runx3 is a target for epigenetic gene silencing already at early stage of gastric carcinogenesis . In the multi - step process of intestinal type gastric carcinoma, genetic instability and hyperplasia of htert positive stem cells precede replication error at the d1s191 locus, dna hypermethylation at the d17s5 locus, ps2 loss, rar - beta2 loss, runx3 loss, abnormal transcripts of cd44 and p53 mutation . All of these changes accumulate in at least 30% of incomplete intestinal metaplasia and are common events in intestinal type gastric cancer . An adenoma to carcinoma sequence is observed on around 20% of gastric adenoma with apc mutations . Molecular events associated with this sequence are loss of heterozygosity and mutation of p53, reduced p27 expression, loss of runx3, over - expression of cycline e and abnormal c - met transcription . The resulting advanced intestinal type gastric carcinomas frequently exhibit dcc loss, apc mutation, 1q loh, loss of p27, reduced tgf beta - receptor expression, reduced nm23 and c - erbb2 gene amplification [13]. Meanwhile, diffuse type gastric carcinogenesis involves loh at chromosome 17p, loh or mutation of p53, lunx3 loss and mutation or loss of e - cadherin . Gene amplification of k - sam, c - met and cycline e confers progression and metastasis . Several of the above molecular events may be present in mixed gastric cancer that have both intestinal and diffuse components [13]. Meta - analysis of epidemiological studies and animal models shows that both intestinal and diffuse types of gastric cancer are equally associated with h. pylori infection [36, 37]. However, h. pylori infection may play a role only in the initial steps of gastric carcinogenesis . Importantly, younger h. pylori - infected patients have a higher relative risk for gastric cancer than older patients . In general, diffuse type gastric cancers hereditary diffuse gastric cancer caused by germ line e - cadherin mutations is also observed in younger age group . We recently found that a low caga igg titer is a useful biomarker to identify a high - risk of gastric cancer and current smoking exhibits significantly higher risk for diffuse type than intestinal type gastric cancers, while radiation risk is significant only for nonsmokers and diffuse type gastric cancers . Differences in h. pylori strain, patient age, exogenous and endogenous carcinogens and host genetic factors may be implicated in two distinct major genetic pathways for gastric carcinogenesis . Gastric cancer cells express a wide array of growth factors and cytokines that act via autocrine, paracrine and juxtacrine mechanisms in the tumor microenvironment [2, 3]. These complex interactions between tumor cells and stromal cells confer morphogenesis, angiogenesis, invasion and metastasis [fig . 1]. Again 1tumor microenvironment of gastric cancer is composed of interaction between cancer cells and stromal cells via growth factor / cytokine network tumor microenvironment of gastric cancer is composed of interaction between cancer cells and stromal cells via growth factor / cytokine network the egf family including egf, tgf alpha, cripto and amphiregulin (ar) are commonly overexpressed in intestinal type carcinoma . Meanwhile tgf beta, insulin - like growth factor ii (igf ii) and bfgf are predominantly overexpressed in the diffuse type carcinoma . Co - expression of egf / tgf alpha and egfreceptor correlates well with the biological malignancy, as these factors induce metalloproteinase . Reported that gastric cancer cells express neutrophilin-1 (nrp-1), a co - receptor for vegf receptor 2 endothelial cells . Egf induces both nrp-1 and vegf expression, suggesting that regulation of nrp-1 expression in gastric cancer is intimately associated with the egf / egfr system . Il-1 alpha is produced not only by activated macrophages but also by gastric cancer cells . It acts as an autocrine growth factor for gastric carcinoma cells and plays a pivotal role as a trigger for the induction of egf and egf receptor [2, 3]. Serum il-6 in patients with gastric cancer is significantly correlated with tumor stage, depth of tumor invasion, lymphatic invasion, venous invasion and hepatic metastasis, suggesting that il-6 may be useful for a prognostic factor of gastric cancer . Interestingly, lin et al . Recently found that il-6 induces gastric cancer cell invasion via activation of the c - src / rhoa / rock signaling pathway . Overexpression of these cytokines, il-8 and growth factors by the tumor cells may be caused mainly by constitutive nf - kb activation due to constitutive activation of several upstream kinases . More than 80% of primary gastric cancer express il-8 and il-8 receptor and this co - expression correlate directly with tumor vasularity and tumor invasion . Il-8 enhances expression of egf receptor, type iv collagenase (mmp9), vegf and il-8 mrna itself by gastric cancer cells, while reducing e - cadherin mrna expression . Recently reported that the cox-2 pathway might be also involved in il-8 production in gastric cancer cells . In addition to il-8, majority of gastric carcinoma express il-11 and il-11 receptor alpha and il-11 promotes the migration of gastric cancer cells by the activation of the phosphatidylinositol-3 kinase pathway . Moreover, recent studies show that expression of il-12 and il-18 also confers progression and metastasis [47, 48]. The negative growth factor tgf beta is frequently overexpressed in gastric carcinoma, particularly in diffuse type carcinoma with productive fibrosis . Reported that active tgf beta 1 is present in the tumor cells and fibroblasts, and high tumor tgf beta1 activity are significantly associated with worse survival of the patients . More importantly, recent in vivo and in vitro studies of mishra group [50, 51] demonstrate that inactivation of elf / tgf - beta signaling plays a key role in the development of gastric carcinoma . Embryonic liver fodrin (elf) is a beta - spectrin, an adaptor protein that plays an essential role in the propagation of tgf beta signaling . She found that loss of elf and reduced smad4 expression are observed in human gastric cancer . Angiogenic factors such as vegf, bfgf and il-8 are produced by tumor cells and results neovacularisation within gastric carcinoma . Vegf promote mainly angiogenesis and progression of intestinal type gastric cancer while bfgf has a stronger association with diffuse type gastric cancer . Vegf - c produced by tumor cells participates in the development of lymph node metastasis . Stromal cells, especially fibroblasts stimulated by growth factors and cytokines such as il-1 alpha, tgf alpha and tgf beta secrete hepatocyte growth factor / scatter factor (hgf / sf), which functions in a paracrine manner as a morphogen or motogen [2, 3]. Hgf / sf from stromal cells binds to c - met on the tumor cells leading to enhanced progression via activation of c - met pathways . Moreover, keratinocyte growth factor (kgf) produced by gastric fibroblasts binds to kgf receptor, k - sam on tumor cells, resulting in the development of scirrhous type gastric cancer . Osteopontin (opn), also termed eta-1 (early t - lymphocyte activation-1), which is a reported protein ligand of cd44, is overexpressed in 73% of gastric cancer [2, 3]. The co - expression of opn and cd44 v9 in tumor cells correlates with the nodal metastasis in diffuse type gastric cancer . Wu et al . Recently reported that elevated plasma opn level is significantly associated with gastric cancer development, invasive phenotype and survival, suggesting that plasma opn might have potential usefulness as a diagnostic and prognostic factor for gastric cancer . Regenerating islet - derived family, member 4 (reg iv) high reg iv expression is associated with 5-fu resistance in gastric and colon cancer cells by inhibiting the mitochondrial apoptotic pathway, suggesting that expression of reg iv is a marker for prediction of resistance to 5-fu chemotherapy in patients with gastric cancer . Excitingly we found that reg iv as well as mmp-10 are able to be useful for biomarkers to detect early stage of gastric cancer . Moreover, interferon inducible gene 6 - 16, gip3 is also expressed in gastric cancers and inhibits mitochondrial - mediated apoptosis in gastric cancer cells, suggesting that 6 - 16 may be a new target for cancer therapy . Wnt-5a is a representative of wnt proteins that activate the beta - catenin - independent pathway . Wnt-5a inhibits proliferation, migration, and invasiveness in thyroid tumor and colorectal cancer cell lines [58, 59], whereas wnt-5a expression is correlated with cell motility and invasiveness in melanoma cells and breast cancer cells with tumor -associated macrophages [60, 61]. . Showed that expression of wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion . Wnt-5a activates pkc and jnk, thereby leading to the activation of fak and paxillin . Wnt-5a and extracellular matrix bind to frizzled (wnt-5a receptor) and integrin, and wnt-5a may be a novel biomarker of prognostic factor and also a therapeutic target for gastric cancer . These events of abnormal growth factor / cytokine networks in the tumor microenvironment will no doubt provide a great deal of information on new approaches for biomarkers and effective therapeutic targeting of gastric cancer.
Although lee had evinced curiosity about the natural world and a desire to understand it as a child, he did not focus in high school on building a strong precollege rsum (hartwell, 2002b) and so began his further education at glendale junior college . However, his academic interests had been awakened by then, and his abilities had begun to show, so he was able to transfer to caltech after just one year . While at caltech, he spent much of his time on independent reading courses and research . He was influenced particularly by work with bob edgar on phage t4 morphogenesis (hartwell, 1961), which gave him a strong belief in the value of conditional mutants for elucidating the organization of essential cellular pathways . After a quick phd in bacterial biochemical genetics with boris magasanik at mit (hartwell and magasanik, 1963, 1964) and an even quicker postdoc with renato dulbecco and marguerite vogt at the salk institute (dulbecco et al ., 1965; hartwell et al ., 1965), he began his independent career in 1965 at the newly established university of california irvine campus . Although he originally intended to study cell - proliferation control and cancer in mammalian cells (and secured a national institutes of health grant for this purpose), he quickly realized that fundamental understanding would come more easily in a system with genetic tools, and he began to work on yeast with lots of early help from bob mortimer in berkeley and herschel roman and don hawthorne in seattle . True to lee s early imprinting in the edgar lab, he began by isolating a large collection of temperature - sensitive lethal (ts) mutants and screening them for defects in macromolecule synthesis, morphology, and/or cell division (hartwell, 1967). Although he was interested from the beginning in using the mutants to learn about the cell cycle, pursuit of this goal was initially blocked by 1) the lack of a way to recognize cell - cycle mutants among the many defective in other essential cellular processes and 2) the near impossibility, at the time, of identifying the molecular defect in any cell - cycle mutant that was identified . (mutants with specific defects in dna replication provided an early exception to both problems . The general solution to problem 2 did not appear until some years later, when the cloning of genes by complementation of the mutations became possible [nasmyth and reed, 1980].) Thus, lee partnered with his uc irvine colleague cal mclaughlin in a series of studies of mutants with specific defects in rna or protein synthesis (hartwell and mclaughlin, 1968; hartwell et al ., in the midst of this period, herschel roman succeeded in convincing lee to move from irvine to the department of genetics at the university of washington; he arrived in the fall of 1968 unencumbered by lab members, except for one student who had finished his experimental work at irvine and was writing his thesis . Mary ashton was hired and began unpacking boxes several weeks before lee himself arrived . By september 1970, the group had grown to eight full - time members (lee, one technician, two postdocs, and four graduate students) and stayed that size during the next four years . Cal mclaughlin, at irvine, continued to isolate many more ts - lethal mutants and send those with possible cell - cycle defects on to seattle for further analysis . Although the group was not large, it never seemed small, in part because interaction among the lab members was so constant and intense, and in part because we were embedded in a highly interactive department of other small groups . One lab room was shared with the group of walt fangman, frequently bringing him and his lab members tom petes, carol newlon, and walter hill into the conversations; the lab of collaborator breck byers was in the adjacent hallway; and the whole department attended weekly seminars and student / postdoc journal clubs, while all the yeast workers (from multiple laboratories) attended the weekly yeast meeting . Herschel himself missed none of these events, took care to ask frequent (and often seemingly nave) questions in order to establish an atmosphere of open and uninhibited inquiry, and left no doubt whatsoever that everyone else s attendance and intellectual engagement at these events was expected . Lee himself was there all the time, doing his own experiments and discussing our experiments and ideas with us on a daily basis . He never joined in our silliest pursuits (nerf and pipette - bucket basketball brought only a half - resigned shake of his head) but otherwise was part of the gang, even for much of our frequent out - of - the - lab socializing . Our scientific ideas did not always bring instant approval indeed, sometimes lee would give only a blank look and wander away without comment . But he would usually return to the idea later without further prompting, either to point out a fatal flaw or to provide a major conceptual improvement and/or suggest a new critical experiment . Lee s leadership style plus (we suppose) a fortunate confluence of personalities led to an atmosphere of total openness and high collegiality, in which no one worried about who would get credit for a particular idea . This was fortunate, because with the constant production, group dissection, and replacement of our often half - cocked models, it was usually impossible to reconstruct exactly who had contributed what facet of a new idea . Each new result generated vigorous discussions so that it is impossible to know who contributed a particular idea (hartwell, 1993); virtually identical wording appears in the acknowledgments section of brian s phd thesis . Importantly, we were excited about what we were doing and believed in its significance, even if others did not (see also below), and we certainly did not worry about what journals our papers would be published in or how many citations those papers would get in the next two years! Brian had begun college in 1965 with an interest in biology, but he was majoring in chemistry because he had found the biology courses boring . Dave stadler s genetics course in the spring of 1968 gave him new hope, so he inquired that fall about research opportunities in the department of genetics . He was directed to the newly arrived lee, who put him to work on a series of highly imaginative experiments that produced no useful results, while also giving him lots of one - on - one teaching about the important processes that had been elucidated using genetic approaches . One of the unproductive studies was an attempt to see whether mutants in the ts - lethal collection that made abnormal - shaped buds (thought of at the time as cell - wall mutants; later found to be defective in the septin proteins and thus in cytokinesis) would pass that trait on to their progeny after return to permissive temperature, as in the cortical inheritance that tracy sonneborn had described in paramecium . On the evening of november 30, 1968, while examining the time - lapse experiments that brian was doing for this project, he and lee suddenly realized that the progressive growth in bud size during the cell cycle allowed them to recognize mutants that were competent for growth but defective in cell division . Establishing the pattern for the lab (see above), neither of them could remember afterward exactly how the idea had developed between them on that eureka! Evening and in the following months . However, by june 1969, it had progressed to the point that brian spent most of the summer screening (with mary ashton s help) the hundreds of ts mutants by time - lapse microscopy at 37c, a project that was rather painful because the lack of a better method at the time forced them to do the screening while sitting and sweating at a microscope in the warm room . By the time brian left to start graduate school at mit in september, they had identified 150 mutants with putative defects in cell division (rather loosely defined). Brian lasted less than a year at mit before the lure of the cell - division mutants (and of the washington mountains) drew him back to seattle and the hartwell lab; the activation energy for this move was lowered by lee s having during the year acquired a warm - air blower and a plexiglas box that fit over the microscope, so time - lapse analyses of the mutants at 37c no longer needed to be done in the warm room! (lee s discovery that conventional window screening provided a grid of just the right size to allow repeated relocation of the same field for time - lapse observations was another critical, high - tech advance .) Fortunately, herschel had saved a slot on the training grant for just such an eventuality . Brian immediately resumed his role as an indefatigable discussant of ideas in the lab and solidified the idea of start (see below) by using the mutants to show that mating was only possible in g1 before cells had initiated the cell cycle; the publication of this work (reid and hartwell, 1977) was held up by brian s injudiciously beginning medical school in the fall of 1975 . Although joe had also begun (as a freshman) at uc irvine in 1965, he and lee did not cross paths until a meeting in the summer of 1968 that almost ended joe s career before it began . While doing undergraduate research in cal mclaughlin s lab, joe ran out of yeast medium and borrowed a flask of medium from lee s lab . It happened to be the last such flask, and lee needed it for an experiment in progress that thus needed to be aborted . Fortunately, his initial anger subsided when he recognized an innocent mistake by an eager student, and he responded positively when joe contacted him later that year about coming to seattle to do graduate work in his lab . Indeed, perhaps anxious that his tiny group would dwindle to nothing when brian left, lee suggested that joe come early . Joe arrived on a weekend day in june, found brian in the lab, and began a conversation that continued almost nonstop for the next five years . Joe s arrival was propitious, because although brian and lee had identified many presumptive cell - division mutants by then, they did not really know whether these were cell - cycle mutants, because they had not yet been able to stain the nuclei in order to assess the status of the nuclear cycle . Brian mentioned this problem to joe in their initial conversation, and joe took up the challenge after only a short delay occasioned by a different (and now forgotten) project suggested by lee that went nowhere . Joe was already making some headway when lee learned that master cytologist carl robinow (matile et al ., 1969) was coming to seattle for a conference and asked him to show joe how to optimize his staining . Joe spent a month gathering and preparing the reagents for robinow s staining protocol, but then forgot to start a culture of cells the night before the scheduled demonstration! Fortunately, he was able to borrow someone else s culture, professor robinow (a proper gentleman in a tweed suit) was able to overcome his instinctive aversion to joe s seattle hippie sartorial style (joe still remembers robinow s big sigh upon their first meeting at the hotel), the demonstration was duly made, and joe s giemsa staining of nuclei improved significantly . This immediately led brian and lee to conclude (in a closed - door meeting that joe remembers well but brian does not) that all the tools were now in place to declare a focus on the cell cycle, a decision whose rationale was explained to joe and the lab helpers over pizza and beer at the northlake tavern (one of many key scientific conferences to occur at that conveniently located and still operational establishment). Nuclear staining using the robinow procedure contributed importantly to all of the earliest papers on the cdc mutants (hartwell, 1971a, b; hartwell et al . In late 1969 or early 1970, joe and lee also began the first attempts to use the mutants to organize the events of the cell cycle into pathways . There was not much to go on at first (too few mutants and too little information about them), and the first model was later shown to be largely incorrect . However, the effort continued more or less nonstop, particularly after brian returned from mit to help drive the frequent conversations . At various times, the models incorporated clocks, stopwatches, dependent series of events, events in independent pathways, and feedback loops that could enforce event dependencies . Such a feedback loop was present even in the very first model and was the forerunner of the now - famous cell - cycle checkpoints, but at the time we had neither a clear concept about how such feedbacks might work nor any experimental handle on them . Thus, the first model that we presented to the world offered no explanation for why some events would be dependent upon earlier events (hartwell et al ., 1974) and implicitly presumed that the progression of the cell cycle could be thought of like the sequential assembly of components during phage t4 assembly (the early edgar influence again). Thus, the proper development of the checkpoint idea was delayed until ted weinert joined lee s lab long after the rest of us had gone (weinert and hartwell, 1988; hartwell and weinert, 1989). We did worry in 1973 that the hypothesis that bud emergence and nuclear migration were in a pathway independent from that including dna replication and nuclear division (hartwell et al ., 1974), which was based solely on the continuation of the nuclear cycle in the cdc24 mutant, might be wrong if that mutant were missing a control that would normally slow or stop the nuclear cycle if bud formation were interrupted . It was this worry that led john to begin focusing on cdc24 and similar mutants several years later in his own lab . However, this focus was ultimately more revealing about the mechanisms of cell polarization than about cell - cycle pathways per se, and it was many years later that danny lew and steve reed (himself a former hartwell - lab postdoc) showed that there is indeed a morphogenesis checkpoint that normally delays mitosis when budding is defective (lew and reed, 1995; short, 2015). This checkpoint was apparently defective in the heavily mutagenized cdc24 strain that was examined in the original studies . In the days before genome sequences, the mapping of a gene gave it an aura of solid reality and helped to prove that it really was distinct from the other genes under study . In the early 1970s, it was also a time - consuming and often frustrating business . Thus, the hartwell group was very lucky that bob mortimer came to spend a sabbatical with don hawthorne in 1972 and used the new cdc mutants to test some new mapping methods . Thus, the mapping of 14 cdc genes to distinct chromosomal positions was reported in the paper summarizing the genetic analysis of the mutants defining genes cdc1cdc32 (hartwell et al ., 1973). Rereading this paper many years after its publication reminded us of the craftsmanship that complemented the creativity: despite the brutally heavy mutagenesis that had been used (hartwell, 1967), the mutations in the many mutants were neatly sorted into genes by a combination of complementation tests, linkage analysis, and mapping; the mutants carrying more than one relevant mutation were identified; and so on . Only minor tweaking of this initial analysis was required later . John had fallen in love with yeast, genetics, and cell biology during required courses as a first - year graduate student at harvard . Undeterred by the complete absence of labs working on yeast in the boston area in 1964, he began thesis work on some yeast proteins with protein chemist guido guidotti while plotting a path to seattle, which at the time had the only concentration of yeast geneticists in the country . In 1968, herschel agreed to sponsor him to come to the department but immediately began nudging him toward the lab of the newly recruited lee . John initially resisted, because he was not excited about the work on rna and protein synthesis, but when he heard about the cell - cycle project from lee and joe (brian was already gone) during a visit in september 1969, he was immediately hooked and signed up on the spot to become lee s first postdoc . After arriving to stay in july 1970, john was trying to decide which cdc mutants to work on when he got distracted by the serendipitous observation that some cultures that had overgrown to stationary phase contained almost exclusively unbudded cells that varied enormously in size . These observations immediately suggested the existence of a control point in the unbudded (g1) phase beyond which cells would complete a cell cycle even if nutrients were not sufficient for normal bud growth . Lee was initially uninterested, in part because herschel argued in yeast meeting that this behavior was strain specific and thus not of general interest . However, lee had already begun using a sonicator to separate clumps of cells before counting with the coulter counter, and when john used this approach to separate postdivision cells in cultures of various strains that had been limited for carbon and energy, nitrogen, or sulfur, he found that the unbudded, size - heterogeneous arrest was quite general . Lee s interest was now awakened, and he politely vacated his office for hours at a time (to be sure, it also gave him a good excuse to do experiments rather than push paper!) While john worked in there with the lights off to do the critical experiment that established the cell - size requirement for cell - cycle initiation . (true to the low - tech spirit of the time, this involved using a filmstrip projector to project individual images from a time - lapse series of stationary - phase cells beginning to grow again onto a piece of white paper taped to the office wall, measuring the long and short axes of the cells with a ruler, and calculating their volumes using the formula for a prolate ellipsoid . Lee also made a critical experimental and conceptual contribution by showing (in collaboration with tom manney and wolfgang duntze) that the response of mating - type a cells to the mating pheromone from cells could also be explained by a similar control point in g1 (bcking - throm et al . He also recognized that we, needed a name for this putative control step, which prevented cell - cycle initiation until certain threshold conditions had been met . He initially suggested sisyphier (after the mythological sisyphus, whose fate was to eternally try, and fail, to push his rock to the top of the hill), but this suggestion (which was perhaps not entirely serious) was quickly rejected, because we did not have any evidence that the cells actually approached cell - cycle initiation but then regressed . Extensive further discussions in the lab and at the northlake then led to the highly imaginative start, which has endured . Meanwhile, lynna had joined the genetics department and the hartwell lab in september 1970 and was contributing in multiple ways . Gee whiz to her, and she posted a map of the london underground system to spoof our early maps of cell - cycle pathways) helped us to sharpen our thinking . And although she preferred to work on the more concrete issue of dna replication (hereford and hartwell, 1971, 1973), her most enduring contribution (probably to her chagrin) was to solidify the concept of start by using epistasis analysis and the newly invented reciprocal - shift method to establish an order of dependent events (start [defined by the cdc28 mutation and the mating - pheromone - sensitive step] cdc4 function cdc7 function) leading up to the initiation of dna replication (hereford and hartwell, 1974). This order of events was further solidified by the work of breck byers and loretta goetsch in the next hallway, who showed that these steps also corresponded to discrete stages in the duplication and separation of the spindle - pole body (byers and goetsch, 1974, 1975). Although lee s brilliance was certainly recognized early (e.g., by hershel s recruitment of him in 1968 and the eli lilly award from the american society for microbiology in 1973), the cell - cycle analysis did not immediately take the world by storm . Although it seems absurd in hindsight, at the time many doubted that yeast was even a bona fide eukaryote, much less that anything useful could be learned about animal cells by studying the evolutionarily distant yeast . An early seminar by lee in vancouver was attended almost exclusively by people from seattle who drove up to hear him, and after a talk at caltech, max delbrck commented that he did not expect to understand the cell cycle any better than he did black holes . And when joe began his postdoc at caltech and was asked at a dinner party to describe his graduate work, the entire party broke down in laughter when he suggested that his work in yeast might someday be relevant to understanding cancer! However, we had all benefited from the diverse faculty that herschel had assembled in the genetics department, and we were aware of the emerging theory that cancer was a disease of clonal evolution, largely because of work in stan gartler s laboratory directly above our own (linder and gartler, 1965). Decades later, when brian told another cancer researcher that he had decided during graduate school to study cancer because of insights into start and cell - cycle control that came from the early yeast investigations, he got a surprised look and the comment that you must have been the only one who thought that then . Of course, it is now abundantly clear that the cdc genes have had the last laugh . Thirty - two such genes were described in the 19701974 papers, and an additional three were described in joe s dissertation, although not in the papers . With only a few exceptions, these original 35 cdc genes have had major impacts on understanding of nearly all aspects of the cell cycle, not only in yeast but also in other organisms (table 1). In most cases, the official names of the yeast genes are still the original cdc names, indicative of how the path to current understanding really did begin with the original cdc mutant . Many other cdc genes were named later, and some have also proved to be quite important in understanding cellular function in both yeast and animal cells, but cdc1cdc35 set a very high bar for subsequent studies! The 35 original cdc genes today, and their human homologues . Cdc name (with current standard name, where different, in parentheses); entries link to the saccharomyces genome database (sgd) (pmid: 22110037). Summarized from information provided in the sgd entries . As approved by the hugo gene nomenclature committee . E - values were obtained by querying the ncbi human taxon (taxid:9606), restricted, nonredundant protein sequence data set with the predicted amino acid sequence of the yeast genes using blastp . Summarized from the literature; see the supplemental material for details and references . Cdc13 and pot1 do not have detectable sequence similarity but are considered to be structurally similar (see the supplemental material for relevant references). The morals of this story are perhaps obvious, but we emphasize some of them here because of their relevance to the current troubled times in academic science . Indeed, we suggest (on the basis of this and many other examples) that it probably flourishes best when the group is small and the lab head is around most of the time and participating actively in the intellectual and experimental life of all group members on an almost daily basis . Second, such research benefits from an atmosphere in which all group members share new results and ideas freely with one another and without concern about the ultimate distribution of credit . Indeed, it is helpful when this attitude can be extended beyond the immediate group to others, as illustrated in this story by the critical early contributions of don hawthorne, carl robinow, bob mortimer, tom manney, wolfgang duntze, and breck byers . Third, if a research group is doing something that excites them, it does not really matter whether anyone else yet realizes that their line of work is important . Indeed, it is better if they do not, because that reduces any possible worries about being scooped, encourages the full development of stories rather than the publication of half - baked ones, and suggests that what the group is doing is really new, rather than yet another variation on a well established theme . These consequences of genuine novelty are, of course, the fundamental and fatal flaw of any measure of scientific accomplishment that uses citation counts, particularly from the first few years after publication . Finally, despite the lack of respect they get from most current grant - review panels, fishing expeditionsin which one does not actually know beforehand what will be discovered are often the most productive form of research, as not only the cdc story but many others clearly show.
Raised blood pressure (bp) is common after acute stroke, whether of ischaemic or haemorrhagic type . It exists in more than three quarters of patients, of which about half have a history of hypertension, and it declines spontaneously in two - thirds of cases returning to prestroke levels over the first week . Its decrease usually occurs 410 days after stroke, but in a significant percentage of patients it falls by about 2530% just within the first 24 hours; particularly when they are moved to a quiet room, they are allowed to rest and their bladder is empty . Mechanisms and effects of elevated bp in this clinical setting have not been well understood . It might be attributable to either one more of the following conditions: preexisting, inadequately treated or undiagnosed hypertension, stress of hospitalization, raised intracranial pressure, haematoma expansion, damage to autonomic centers and abnormal baroceptor sensitivity, neuroendocrine response with activation of sympathetic nervous system, renin - angiotensin axis and/or glucocorticoid system, and myocardial changes [36]. Most of the studies, although not all, have found that high bp in the acute phase of stroke, whether measured as casual or 24 hours ambulatory readings, is associated with a poor outcome [79] and an increased risk of death and dependency [1014]; a u - shaped relationship between bp values and outcome has been described in different studies [1517]. Recent evidence suggests that not only bp but even its derived indices and other haemodynamic measures as mid blood pressure, mean arterial pressure, bp variability, heart rate, pulse pressure, and rate - pressure product are related to functional outcome [1820]. The association is thought to be related to the early stroke recurrence and the development of cerebral edema and greater serious haemorrhagic transformation in ischaemic stroke [21, 22] and to the haematoma expansion in primary intracerebral haemorrhage . While observational studies show that high bp is independently associated with a poor outcome, suggesting that it should be lowered, pathophysiology argues that lowering bp will reduce cerebral blood flow when cerebral autoregulatory mechanisms are impaired . Additionally, in acute ischaemic stroke the infarcted brain tissue may be surrounded by a penumbra zone of underperfused but viable tissue where cerebral blood flow extremely depends on the systemic bp and collaterals until the occluded artery is recanalized . Lowering bp carries the risk of jeopardizing the perfusion of this area leading to an increase of brain infarction or perihematoma ischaemia . Spontaneous thrombolysis may also occur, and the ischaemic area may become hyperaemic; at this stage a very high bp might cause propagation of infarct - related brain oedema or haemorrhagic transformation of the infarct . Unfortunately there is no sure clinical correlate of spontaneous thrombolysis, and in routine clinical care it is not possible to judge when it is better to leave a very high bp untreated or when it is necessary to intervene . In summary, there is still debate in whether, when and how high bp should be lowered (epidemiological evidence) or not (pathophysiological concerns). Different antihypertensive drug classes might have differential effects considering both their action in lowering bp and specific organ effects: for example -blockers might be detrimental, and the use of calcium channel blockers was associated with a worsening of outcome in some studies, especially those testing intravenous formulation, perhaps because of a reduction in cerebral perfusion . Our purpose is to investigate through a research of the recent literature the effect of the angiotensin receptor blockers (arbs) administered in the acute phase of stroke on death and dependency . We used medline (1985 to december 2011; any language) to identify randomized controlled trials of arbs in patients within 72 hours of stroke . We selected trials of more than 100 patients which assessed the effect on death or dependency and recorded coronary heart disease events or stroke (irrespective of whether bp lowering was considered the mechanism of action). Search terms were blood pressure lowering, blood pressure reduction, antihypertensive, or hypertension or receptor, angiotensin / antagonists and inhibitors or the name of all arbs listed in the british national formulary as keywords or text words . We also searched the cochrane collaboration and web of science databases and the citations in trials and meta - analysis . Randomized trials were included irrespective of participants' age, disease status, bp before treatment, and the use of other drugs . Confounded trials (in which 2 active treatment were compared in the absence of a control arm) were excluded . The authors extracted data independently from identified publications with respect to patients' number, sex, age, previous medical history, stroke subtype, time from stroke to enrollment, baseline bp, difference in bp between treatment and control groups, follow up period, grade of dependency, and death . Outcome events included stroke (all, fatal, and nonfatal), myocardial infarction (all), total vascular events (combined stroke, mi, and vascular death), and mortality (all - cause, vascular). Three randomized placebo - controlled multicenter studies fulfilled the inclusion criteria (table 1), each of which had been published [2729]. The combined sample size was 3728 with almost two - thirds of the data coming from one study (scast). In two studies (access and profess) all patients recruited had ischaemic stroke, while in the scast trial 85.4% of patients had ischaemic and 13.5% haemorrhagic stroke; mean time from stroke to enrollment varied from 17.6 to 57.6 hours, and recruitment was limited to patients with high bp . Main characteristics of each trial are summarized in table 2 . In the access study, protocol considered a 7-day placebo controlled phase: treatment was started with 4 mg candesartan cilexetil daily or placebo on day 1; on day 2, dosage was increased to 8 or 16 mg candesartan cilexitil or placebo if bp exceeded 160 mmhg systolic or 100 mmhg diastolic; target was a 1015% bp reduction within 24 hours . On day 7, a 24-hour bp profile was obtained in all patients: in those in the candesartan cilexitil group who showed a hypertensive profile (mean daytime bp> 135/85 mmhg) dosage was increased or an additional antihypertensive drug was added; in placebo arm, candesartan cilexitil was started in patients with hypertensive profile to lower bp to <140/90 mmhg (office bp) or <135/85 mmhg (mean daytime bp), while those with a normotensive profile did not receive antihypertensive medication . Follow - up examinations were performed after 3, 6, and 12 months . The profess study compared the effect of telmisartan (80 mg daily) versus placebo and combined aspirin (25 mg twice daily) and extended release dipyridamole (200 mg daily) versus clopidogrel (75 mg daily) in a 2 2 factorial design in patients with recent ischaemic stroke followed up for a mean duration of 30 months . The subgroup analysis reviewed included only patients randomized within 72 hours of stroke onset, evaluated at 1 and 3 months . In the scast trial, patients with diagnosis of stroke (ischaemic or haemorrhagic) presenting within 30 hours of symptom onset were allocated in a 1: 1 ratio to treatment with candesartan or placebo . There was a fixed - dose escalation scheme: 4 mg on day 1, 8 mg on day 2 and 16 mg on days 37 . Subsequent evaluations took place on day 7 and at 1 and 6 months; to avoid important differences in treatment during followup, candesartan was the advised antihypertensive agent and was provided free of charge . In each trial there were no significant differences regarding the use of concomitant medication on hospital admission or during follow - up between the active treatment and placebo groups . In the access trial mean bp on hospital admission was 198/103 and on study onset 189/99; baseline severity of stroke was not directly reported but patients had a mean barthel index (bi) of 62; no data are available about etiopathogenesis of strokes . In the profess subgroup analysis mean baseline bp was 147/84 mmhg and mean national institutes of health stroke scale (nihss) at admission was 3 . According to toast classification, strokes were due to small artery occlusion in 59.5%, large - artery atherosclerosis in 20.9%, cardioembolism in 1.4%, and to undetermined or other determined etiologies in the remaining cases . In the scast study mean baseline bp was 171/90 mmhg and patients enrolled had a mean scandinavian stroke scale (sss) score of 41, equivalent to a nihss of 8 . Strokes have been described following topographic rather than etiological criteria: according to oxfordshire community stroke project (ocsp) classification, strokes were distributed as total anterior syndrome in 8%, partial anterior syndrome in 48.7%, posterior syndrome in 14%, lacunar syndrome in 28.9%, unknown in <1% . Main baseline findings of the included studies are illustrated in table 3 . In the access population no significant difference in bp was evident between the groups during the placebo - controlled phase and in the subsequent followup; at 7 days mean bp was 160/87 mmhg, at 3-months 150/85 mmhg and at 12 months 147/83 mmhg . Only two patients in the placebo arm had a normotensive profile on day 7 and did not receive the antihypertensive drug . In the profess subgroup analysis, telmisartan lowered significantly systolic bp (sbp) by 6 to 7 mmhg and diastolic bp (dbp) by 2 to 4 mmhg over the whole study course: on day 7, bp was135.3 (17.8)/78.4 (10.8) mmhg in treatment arm and 141.4 (17.0)/81.6 (11.0) mmhg in placebo group, with a difference of 6.1/3.2 mmhg (p <0.0001); on day 30, mean bp was 135.7/79.6 mmhg versus 142.6/83.1 mmhg (treatment versus placebo) with a difference of 6.9/3.6 mmhg (p <0.0001); on day 90, mean sbp was 134.5 (19.9) mmhg (treatment) versus 140.3 (19.0) (placebo) with a difference of 5.8 mmhg (p <0.0001) and mean dbp was 79.2 (11.1) mmhg versus 81.5 (11.2) mmhg (treatment versus placebo) with a mean difference of 2.4 mmhg (p = 0.0002). In the scast trial bp fell in both groups during treatment but was significantly lower in patients allocated candesartan than in those on placebo (p 0.001 for days 27); on day 7, mean bp was 147/82 mmhg (sd 23/14) in the candesartan group and 152/84 mmhg (sd 22/14) in the placebo group . The mean difference in sbp on day 7 was 5 mmhg (95% ci 37; p <0.0001) and the mean difference in dbp was 2 mmhg (13; p = 0.001). During the 6-month followup, mean bp values were similar in the two groups, and at 6 months the mean bp was 143/81 mmhg in both groups . In the access the bi revealed no significant difference on study onset and after 3 months (candesartan cilexetil versus placebo, day 0: 60.0 30.2 versus 64.1 27.5; 3 months: 87.0 22.9 versus 88.9 19.9). The cumulative 12-month mortality (candesartan cilexetil versus placebo: 2.9% versus 7.2%; p = 0.07) and the number of vascular events (candesartan cilexetil versus placebo: 9.8% versus 18.7%; p = 0.026) differed significantly in favor of the candesartan cilexetil group; the odds ratio was 0.475 (95% ci, 0.252 to 0.895). The clinical benefit was independent of bp values and mainly attributable to a lower incidence of myocardial ischaemic events . Drug tolerance and number or type of undesirable effects did not differ significantly between the groups . In the profess subgroup analysis combined death or dependency (mrs at 30 days, with adjustment for baseline covariates) did not differ whether analyzed as an ordinal outcome (ordered mrs categories: 0, 1, 2, 3, 46 to maintain proportionality) (or, 1.03; 95% ci, 0.841.26; p = 0.81) or with dichotomization of the data at the median (mrs 0 - 1 versus 26; or, 1.00; 95% ci, 0.771.29). There was no significant difference between the treatment groups for the distribution of ordinal stroke events (fatal, dependent (mrs 25), independent (mrs 0,1), tia, none), for the time to recurrence (p = 0.40) and, similarly, for other events (i.e., death, stroke recurrence, mi, and combined vascular events) at 7, 30, or 90 days . The analysis of the first co - primary effect variable, the cumulative risk of the composite endpoint of vascular death, stroke or myocardial infarction, showed no significant difference between candesartan and placebo (unadjusted analysis hr 1.09, 95% ci 0.841.41; p = 0.53; adjusted analysis hr 1.09, 0.841.41; p = 0.52; per - protocol analysis hr 1.11, 0.851.46; p = 0.46). Regarding the second co - primary effect variable, the functional outcome at 6 months, no significant difference was seen across the mrs categories (unadjusted ordinal regression analysis, or 1.13, 95% ci 0.971.32; p = 0.12); similar results have been obtained in both the fixed dichotomy (mrs 36 versus 02) analysis (unfavourable outcomes in 35% of patients on candesartan and in 33% of patients allocated on placebo; or 1.12, 0.901.41, p = 0.32; rr 1.06, 0.931.19, p = 0.39) and the sliding dichotomy analysis using the sss scores at baseline (unfavourable outcomes in 56% of patients on candesartan and 52% of patients on placebo; or 1.16, 95% ci 0.971.38, p = 0.11; rr 1.07, 95% ci 0.991.16, p = 0.11). For all the secondary outcomes assessed there were no significant differences between treatment and placebo: for all events (death from any cause, vascular death, ischaemic stroke, haemorrhagic stroke, all strokes, myocardial infarction, stroke progression, symptomatic hypotension, renal failure, and symptomatic venous thromboembolism: rr 1.47, 95% ci 1.012.13; p = 0.04), for sss score at 7 days (p = 0.13) and for bi at 6 months (0.47). There were no significant differences between the groups for the adverse events reported by the investigators . Significant differences in bp values between treatment and placebo were therefore found in the profess and scast trials, but in only the first it remained significant during the follow - up; conversely, in the access study a significant difference was never found . In all trials no effect of the active treatment was seen on dependency, death and vascular events at one, three or six months; in the access study a decrease in cumulative mortality and number of vascular, mainly myocardial, events emerged in candesartan group at 12 months . While the primary prevention of stroke through the treatment of hypertension is well established and evidence from randomized controlled trials suggests the use of antihypertensive agents for the secondary prevention of vascular events in patients with previous stroke or transient ischaemic attack, the management of bp immediately after stroke has been an enigmatic controversy for more than two decades, exemplified by a debate published back in 1985, and still remains uncertain . In experimental studies on rats, candesartan has been shown to be neuroprotective with both reduction of neurovascular damage, demonstrated by decreased infarct size, hemoglobin content and oedema in the ischaemic brain hemisphere, and improvement of neurological outcome . These favorable effects were evident with an early post ischemic stroke drug administration, at doses that did not affect or moderately lowered bp [32, 33], suggesting a multimodal protective effect, partly due to bp lowering and partly due to pleiotropic, vascular and neuronal, actions . Homeostatic defense processes against postischaemic brain damage, or almost a part of them, are pressure dependent; presumably there are feedbacks and fine interrelationships among bp, the processes limiting the infarct size and the mechanisms involved in the recovery of ischemic tissue . On the other side, competitive inhibition of the binding of angiotensin (at) ii to at1 receptors allows an unopposed activation of the at2 receptors which has been hypothesized to be protective in focal cerebral ischaemia being responsible of several events as recruitment of cerebral collaterals, normalization of cerebrovascular autoregulation, enhancement of neuronal resistance to anoxia, inhibition of inducible nitric oxide synthetase, reduction of oxidative damage, prevention of apoptosis, promotion of angiogenesis and attenuation of inflammation, endothelial disfunction and prothrombosis [34, 35]. Translating these experimental findings to human stroke patients is quite difficult . The clinician who faces a patient in the acute phase of stroke with elevated bp has these main alternatives: he may choose to continue or not a preexisting antihypertensive drug or to introduce or not a new one . In the continue or stop post - stroke antihypertensives collaborative study, continuation compared with cessation of preexisting antihypertensive drugs for a two weeks period after acute stroke was not associated with a substantial reduction in two - week death or dependency, cardiovascular event rate or mortality at six months . Besides, clinical trials evaluating the effects of different bp lowering drugs administered in the acute phase of stroke have given conflicting results in respect of functional outcome, and the evidence to guide the practicing clinician is still rudimentary . Sharp reduction in arterial bp has been sought to determine a worse short and long - term prognosis [37, 38], while a moderate and caution reduction might be safe and even improve long term mortality and reduce recurrent vascular events [39, 40]. Not only the degree of bp reduction but even the nature of the pharmacological agent itself, considering its specific mechanism of action and in the perspective of possible drug - class related benefits, has been and still remain matter of debate . The purpose of this systematic review is to investigate the role of a specific class drug, the arbs, early administered after stroke . None of the included studies demonstrated a significant benefit of active treatment on functional outcome and stroke recurrence at short and medium term, but some evaluations should be taken in account . Firstly, the mean bp at enrollment was much higher in the access study (189/99 mmhg) in respect to both the profess and scast trials (147/84 mmhg and 171/90 mmhg, resp . ). Secondly, relevant differences existed in stroke severity: patients enrolled in the profess had very mild stroke in respect to those participating to the scast and above all to the access who had a significantly greater impairment . Thirdly, the effect of treatment on bp: candesartan did not alter bp in the access while in the profess, despite the fact that bp at baseline was reasonably well controlled, telmisartan further reduced it; also in the scast trial bp fell in both treatment and placebo but was significantly lower in patients allocated candesartan although difference disappeared during the followup . Finally, the achievement of endpoints: neither functional outcome nor number of vascular events have been positively affected by treatment but in the access study, although the primary outcome (disability at 3 months) was neutral, treatment with candesartan was associated with a significant reduction in secondary outcome including the 12-month mortality and vascular events . Taking into account the u - shaped relationship between bp and outcome in acute stroke, the main advantage deriving from lowering bp treatment would be expected in the access trial which included patients with severely elevated bp . Even in this study, however, 3-month outcome has not been influenced by treatment and the reduction in the number of vascular events observed in favor of the candesartan cilexitil group was mainly due to a lower incidence of myocardial ischaemic events but not of recurrent cerebral ischaemic events which did not significantly contribute to the difference in cardiovascular morbidity and mortality . Much more surprising is the fact that in this study bp has not been affected by treatment, raising the question around the existence of a drug specific effect of the at1 receptor blockade, beyond the hemodynamic activity, able to modulate vascular remodeling and to affect cardiovascular survival with a benefit that did not arise immediately but appeared to increase over the time . Analogous data have been acquired from various cardiac intervention studies suggesting the hypothesis that early neurohumoral inhibition has similar beneficial effects in both cerebral and myocardial ischaemia, although the underlying mechanism is not resolved . These findings, however, have not been confirmed in the profess and scast trials, and such discrepancies may simply reflect a false positive finding of a small trial which was stopped prematurely on the basis of an interim analysis, requiring more investigations . Taking their similarities and differences, the studies we have considered raise doubts over the hypothesis of a specific effect of angiotensin receptors blockade in acute stroke; moreover, conclusions are fully compatible with those of a recent meta - analysis and a regularly update cochrane survey of randomized controlled trials of bp lowering drugs in acute stroke . According to currently available evidence there is no clear evidence of benefit for routine bp lowering treatment in the acute phase of stroke; many pitfalls, however, still remain to explain . Firstly, bp management in this clinical setting may have to be tailored with respect to the underlying etiology, and other parameters than baseline bp may need to taken into consideration [42, 43]. Patients with atherosclerotic or lacunar strokes are often affected by chronic hypertension with subsequent arterial stiffness and shift of the cerebral blood flow autoregulatory curve to the right . Consequently, they may tolerate elevated bp in acute stroke more efficiently, and bp values considered normal for the general population may be inadequate to perfuse the ischaemic brain . These patients usually present also diffuse atherosclerotic lesions in cerebral vessels which compromise the patency of collateral circulation: high bp may be needed to enhance perfusion of the ischaemic penumbra zone . On the contrary, in the cardioembolic strokes patients may only have atrial fibrillation in absence of arterial hypertension or significant atherosclerotic stenosis and may need only moderately elevated bp to promote perfusion through a patent collateral circulation; moreover, cardioembolic strokes tend to be of larger size with a higher risk of edema and hemorrhagic transformation in case of raised levels of bp . Unfortunately, the trials considered neither reported nor stratified outcome data according to the aetiopathogenesis of strokes . Secondly, the interrelationships between baseline bp, stroke severity, and administration of antihypertensive agents have not been well understood but an interaction could not be excluded; in the profess study the failure to show beneficial effect of telmisartan may reflect that patients had only mild hypertension and mild stroke . Thirdly, favourable effect of treatment may show a significant time interaction; a post hoc analysis of the main profess study indicated that recurrence was lower with telmisartan after the first six months of treatment, and in the access benefit on overall mortality and vascular events did not arise immediately but instead appeared to increase during follow up . The included studies presented followup ranging from 3 to 12 months and have not been designed to investigate long term effects of treatment . Although a benefit of early treatment has not emerged, it is noteworthy that a substantial safety has resulted without difference in number or type of undesirable effects in treatment and placebo groups; since chronic lowering bp reduces strokes recurrence [45, 46] it may be safe to start such treatment even acutely in selected subgroups . Many other issues like the timing of starting the treatment, the degree, and rapidity of bp reduction, also taking account of the initial level, and the formulation, route of administration, and doses of different pharmacological agents should be further addressed; it is not possible to rule out that different drugs might have different effects . Firstly, trial - level data rather than individual patients' data were assessed since the latter were not available to us; analyses based on individual patients' data are generally superior and allow subgroup analyses to be performed . Secondly, the inhomogeneity of the patients' population and of the design of the study methods regarding cut offs for bp, stroke severity, time of assessment of the outcome variables; for the profess study, we have considered a subgroup of the patients entered into the main large secondary prevention trial, such that patients' characteristics reflect the inclusion criteria for a study of vascular prophylaxis rather than acute intervention . Thirdly, we could not assess the effect of lowering bp in patients with different subtypes of strokes since trials did not report these data separately . The currently available studies did not identify any clear indication that treatment with the arbs is beneficial in patients with acute stroke and raised bp on functional outcome and stroke recurrence at short and medium term . Many issues, however, should be still considered; two large studies involving more than 2500 patients with acute ischaemic (enos) and haemorrhagic stroke (enos, interact 2) [47, 48] are ongoing, and it is favorable these and future trials will help to clarify many unresolved questions and whether there are subgroups of patients or different approaches to bp management for which a treatment benefit can be obtained.
Cardiovascular diseases (cvd) are the leading cause of death not only in the united states (us) but worldwide. [1 - 7] although cvd mortality has declined consistently and substantially over time, nearly 800,000 deaths still occur annually, accounting for 31% of all deaths in the us. [1 - 5] globally, 17.3 million cvd deaths occur annually, representing 30% of all global deaths . Cardiovascular diseases, including heart disease and stroke, have been the leading cause of death in the us for the last eight decades and contribute more to years of potential life lost than any other disease except cancer . In the united states, the prevalence of smoking, a major risk factor, has decreased markedly over time, which has helped reduce cvd mortality by 75% during the past 60 years . Health inequalities according to racial / ethnic and socioeconomic characteristics have long represented an important area of research in the united states, and reduction of health inequalities, including those in cvd morbidity and mortality, has been an integral part of the national health policy initiative for the past four decades. [1,8 - 11] trends and contemporary patterns in cvd mortality are routinely analyzed by age, sex, race / ethnicity, and place of residence in the united states. [1 - 5] several us studies have also examined differentials in cvd mortality according to socioeconomic status (ses) or area - based deprivation level. [11 - 15] however, studies of time trends in cvd mortality according to area- and individual - level ses are still relatively uncommon in the united states . Previous research has shown substantial and widening socioeconomic disparities in cvd mortality in the us . Research showing increased cvd mortality risks among lower ses groups in the us dates as far back as 1950 . Similarly, historical data showing consistently and markedly higher cvd mortality rates among us blacks than whites and among males than females goes as far back as 1935 . In 2012, the cvd prevalence was at least 50% higher among blacks, hispanics, and american indians / alaska natives than whites; and the cvd mortality rate was 1.3 times higher for blacks than for whites . To our knowledge, comprehensive efforts have not yet been undertaken to examine socioeconomic patterns in cvd mortality across major race - sex groups in a temporal fashion, using annual mortality data for the past four decades . It is important to estimate and monitor the magnitude of cvd mortality disparities across racial and socioeconomic groups for the purposes of setting up cvd prevention and control programs, resource allocation, and target setting . Temporal analysis allows us to assess progress in reducing health inequalities among those at higher risks of cvd morbidity and mortality . It also provides important insights into the role of health - policy and medical interventions in reducing cardiovascular disease burden, such as differential access to health services and availability of drugs / medications to lower cholesterol and blood pressure levels, and surgical treatments, well as of changing socioeconomic conditions and behavioral risk factors such as smoking, obesity, physical inactivity, and unhealthy diet. [6 - 8,10,11] the aim of our study is to examine changes in the extent of socioeconomic and racial disparities in cvd mortality in the us between 1969 and 2013 . We use census - based deprivation indices linked to national vital statistics mortality data to examine area - based socioeconomic differences in cvd mortality over time . Additionally, we use the national longitudinal mortality study (nlms) to analyze temporal variations in cvd mortality risks by individual - level socioeconomic and demographic characteristics . We examine annual trends in cvd mortality among major racial / ethnic groups and analyze whether socioeconomic gradients in mortality vary by sex . To analyze temporal inequalities in cvd mortality, we used the national vital statistics mortality database. [1 - 5,18] since the vital - statistics - based national mortality database lacks reliable ses data, socioeconomic patterns in cvd mortality were derived by linking the 1990 and 2000 census - based county - level deprivation indices to age - sex - race - county - specific mortality statistics from 1969 through 2011. [9 - 11,19,20] we used previously published factor - based deprivation indices from the 1990 and 2000 decennial us censuses . The 1990 deprivation index consisted of 17 census - based socioeconomic indicators, which may be viewed as broadly representing educational opportunities, labor force skills, economic, and housing conditions in a given county . Selected indicators of education, occupation, wealth, income distribution, unemployment, poverty, and housing quality were used to construct the 1990 index . The factor loadings (correlations of indicators with the index) for the 1990 index ranged from 0.92 for 150% of the poverty rate to 0.45 for household plumbing . The 2000 deprivation index consisted of 22 socioeconomic indicators, including five additional measures of income distribution, wealth, and housing quality . The factor loadings for the 2000 index varied from 0.92 for 150% of the poverty rate to 0.39 for household plumbing . The common indicators in the 1990 and 2000 deprivation indices generally had similar factor loadings or relative weights . The correlation between the 1990 and 2000 deprivation indices was 0.97, indicating a fairly stable geographical distribution of deprivation in the us over time . Substantive and methodological details of the us deprivation indices are provided elsewhere . In order to compute mortality rates by deprivation level, we used the weighted population quintile distribution of the deprivation index that classified all 3,141 us counties into 5 groups of approximately equal population size . The groups thus created ranged from being the most - deprived (first quintile) to the least - disadvantaged (fifth quintile) population groups . Each of the 3,141 counties in the mortality database was assigned one of the 5 deprivation quintiles . The 1990 index was used to compute deprivation - specific mortality rates from 1969 to 1998, whereas the 2000 index was used to compute mortality rates by deprivation level from 1999 to 2011 . Cvd mortality rates were computed annually for the total population and by race and sex from 1969 through 2013 . Cvd mortality rates according to county deprivation levels were computed annually between 1969 and 1998 and for the following time periods: 1999 - 2001, 2002 - 2006, and 2007 - 2011 . Our trend analysis included all 41,755,700 cvd deaths that occurred in the us between 1969 and 2013 . Of these, 36,783,348 (or 88.1%) deaths occurred among whites and 4,433,500 (or 10.6%) deaths among blacks . Mortality rates for each area- or individual - level socioeconomic group and race - sex group were age - adjusted by the direct method using the age - composition of the 2000 us population as the standard. [1 - 5,10,11] log - linear regression models were used to estimate annual rates of decrease in cvd mortality for race, sex, and deprivation groups . Specifically, the logarithm of mortality rates were modeled as a linear function of time (calendar year), which yielded annual exponential rates of change in mortality rates . Racial / ethnic and deprivation - specific disparities in mortality were described by rate ratios (relative risks) and rate differences (absolute inequalities), which were tested for statistical significance at the 0.05 level . To examine variations in cvd mortality rates according to individual - level socioeconomic and demographic characteristics the nlms is a longitudinal dataset for examining socioeconomic, occupational, and demographic factors associated with all - cause and cause - specific mortality in the united states . The nlms is conducted by the national heart, lung, and blood institute (national institutes of health [nih]) in collaboration with the us census bureau, the national cancer institute (nih), the national institute on aging (nih), and the national center for health statistics (centers for disease control and prevention). The nlms consists of 30 current population survey (cps) and census cohorts between 1973 and 2002 whose survival (mortality) experiences were studied between 1979 and 2002 . The cps is a sample household and telephone interview survey of the civilian non - institutionalized population in the united states and is conducted by the us census bureau to produce monthly national statistics on unemployment and the labor force . Data from death certificates on the fact of death and cause of death are combined with the socioeconomic and demographic characteristics of the nlms cohorts by means of the national death index . The full nlms consists of approximately 3 million individuals drawn from 30 cps and census cohorts whose mortality experience has been followed from 1979 through 2002, with the total number of deaths during the 23-year follow - up being 341,343 . For this study, we used the 1979 - 1998 nlms data to compute cohort - based mortality rates during 1979 - 1989 and 1990 - 1998 using the person - years approach . Mortality rates in the nlms were expressed as the number of deaths divided by person - years lived, adjusting for age . To analyze temporal inequalities in cvd mortality, we used the national vital statistics mortality database. [1 - 5,18] since the vital - statistics - based national mortality database lacks reliable ses data, socioeconomic patterns in cvd mortality were derived by linking the 1990 and 2000 census - based county - level deprivation indices to age - sex - race - county - specific mortality statistics from 1969 through 2011. [9 - 11,19,20] we used previously published factor - based deprivation indices from the 1990 and 2000 decennial us censuses . The 1990 deprivation index consisted of 17 census - based socioeconomic indicators, which may be viewed as broadly representing educational opportunities, labor force skills, economic, and housing conditions in a given county . Selected indicators of education, occupation, wealth, income distribution, unemployment, poverty, and housing quality were used to construct the 1990 index . The factor loadings (correlations of indicators with the index) for the 1990 index ranged from 0.92 for 150% of the poverty rate to 0.45 for household plumbing . The 2000 deprivation index consisted of 22 socioeconomic indicators, including five additional measures of income distribution, wealth, and housing quality . The factor loadings for the 2000 index varied from 0.92 for 150% of the poverty rate to 0.39 for household plumbing . The common indicators in the 1990 and 2000 deprivation indices generally had similar factor loadings or relative weights . The correlation between the 1990 and 2000 deprivation indices was 0.97, indicating a fairly stable geographical distribution of deprivation in the us over time . Substantive and methodological details of the us deprivation indices are provided elsewhere . In order to compute mortality rates by deprivation level, we used the weighted population quintile distribution of the deprivation index that classified all 3,141 us counties into 5 groups of approximately equal population size . The groups thus created ranged from being the most - deprived (first quintile) to the least - disadvantaged (fifth quintile) population groups . Each of the 3,141 counties in the mortality database was assigned one of the 5 deprivation quintiles . The 1990 index was used to compute deprivation - specific mortality rates from 1969 to 1998, whereas the 2000 index was used to compute mortality rates by deprivation level from 1999 to 2011 . Cvd mortality rates were computed annually for the total population and by race and sex from 1969 through 2013 . Cvd mortality rates according to county deprivation levels were computed annually between 1969 and 1998 and for the following time periods: 1999 - 2001, 2002 - 2006, and 2007 - 2011 . Our trend analysis included all 41,755,700 cvd deaths that occurred in the us between 1969 and 2013 . Of these, 36,783,348 (or 88.1%) deaths occurred among whites and 4,433,500 (or 10.6%) deaths among blacks . Mortality rates for each area- or individual - level socioeconomic group and race - sex group were age - adjusted by the direct method using the age - composition of the 2000 us population as the standard. [1 - 5,10,11] log - linear regression models were used to estimate annual rates of decrease in cvd mortality for race, sex, and deprivation groups . Specifically, the logarithm of mortality rates were modeled as a linear function of time (calendar year), which yielded annual exponential rates of change in mortality rates . Racial / ethnic and deprivation - specific disparities in mortality were described by rate ratios (relative risks) and rate differences (absolute inequalities), which were tested for statistical significance at the 0.05 level . To examine variations in cvd mortality rates according to individual - level socioeconomic and demographic characteristics, we used the 1979 - 1998 nlms data . The nlms is a longitudinal dataset for examining socioeconomic, occupational, and demographic factors associated with all - cause and cause - specific mortality in the united states . The nlms is conducted by the national heart, lung, and blood institute (national institutes of health [nih]) in collaboration with the us census bureau, the national cancer institute (nih), the national institute on aging (nih), and the national center for health statistics (centers for disease control and prevention). The nlms consists of 30 current population survey (cps) and census cohorts between 1973 and 2002 whose survival (mortality) experiences were studied between 1979 and 2002 . The cps is a sample household and telephone interview survey of the civilian non - institutionalized population in the united states and is conducted by the us census bureau to produce monthly national statistics on unemployment and the labor force . Data from death certificates on the fact of death and cause of death are combined with the socioeconomic and demographic characteristics of the nlms cohorts by means of the national death index . The full nlms consists of approximately 3 million individuals drawn from 30 cps and census cohorts whose mortality experience has been followed from 1979 through 2002, with the total number of deaths during the 23-year follow - up being 341,343 . For this study, we used the 1979 - 1998 nlms data to compute cohort - based mortality rates during 1979 - 1989 and 1990 - 1998 using the person - years approach . Mortality rates in the nlms were expressed as the number of deaths divided by person - years lived, adjusting for age . Figure 1 shows changes in relative contributions of cvd and cancer, the top two leading causes of death, to overall us mortality over time . Even though cvd has become relatively less important over time as a cause of death, it still accounted for 31% of all us deaths in 2013 . About 23% of all deaths in 2013 were due to cancer . In 1969, on the other hand, cvd accounted for 53% of all us deaths, and there were 3 times as many deaths from cvd as from cancer . In terms of age - specific risks, cvd is a more prominent cause of death than cancer for infants and at older ages 75 years and beyond, while cvd and cancer exert similar mortality risks between ages 20 and 74 years (figure 1). Relative contribution of cardiovascular disease (cvd) and cancer to overall mortality and age - specific patterns in cvd and cancer mortality, united states, 1969 - 2013 figure 2 presents annual cvd mortality trends from 1969 to 2013 for the total us population and by race / ethnicity and sex . During 1969 - 2013, mortality rates declined consistently between 1990 and 2013 for american indians / alaska natives (aians), asians / pacific islanders (apis), and hispanics at average annual rates of 2.79%, 3.11%, and 2.47%, respectively . Trends in cardiovascular disease (cvd) mortality by race / ethnicity and sex, united states, 1969 - 2013 while absolute black / white differences in cvd mortality, as measured by rate differences, narrowed somewhat during the last two decades, racial disparities in cvd mortality, as measured by rate ratios, widened consistently between 1969 and 2013 (figure 2). Compared to whites, blacks had 12% higher cvd mortality in 1969 but 38% higher mortality in 2007 . In 2013, blacks had 30% higher cvd mortality than whites, but apis, aians, and hispanics 25 - 39% had lower cvd mortality than whites . Area socioeconomic gradients (rate ratios) in cvd mortality increased substantially during 1969 - 2011 (figure 3). Compared to those in the most - affluent group, individuals in the most - deprived area group had 11% higher cvd mortality in 1969, but 40% higher mortality in 2007 - 2011 . During 1969 - 2011, even though cvd mortality rates decreased by at least 62% for all deprivation groups, more deprived groups had higher mortality rates than less deprived groups in each year / time - period (figure 3). Cvd mortality declined at a much faster pace among individuals in the most affluent group than those in more deprived groups . During 1969 - 2011, cvd mortality in the five most - to - least deprived groups decreased at average annual rates of 2.20%, 2.48%, 2.54%, 2.66%, and 2.71%, respectively . Absolute socioeconomic disparities in cvd mortality for the total, male, and female populations, as measured by rate differences between the most and least deprived quintiles, also widened between 1982 and 2012 . Us cardiovascular disease (cvd) mortality by area socioeconomic deprivation index, 1969 - 2011 cvd mortality rates decreased with decreasing deprivation levels for both white and black populations, with socioeconomic gradients in cvd mortality increasing consistently during 1969 - 2011 for both racial groups (data not shown for brevity). Absolute socioeconomic inequalities also widened over time for both whites and blacks . During 2007 - 2011, cvd mortality rates varied from a low of 118.23 deaths per 100,000 population for aians in the least - deprived group to a high of 334.59 for blacks in the most - deprived group . Whites, blacks, aians, apis, and hispanics had, respectively, 1.36, 1.32, 1.76, 1.27, and 1.42 times higher cvd mortality in the most deprived group than in the least deprived group (figure 4). Cardiovascular disease (cvd) mortality by race / ethnicity and socioeconomic deprivation level, united states, 2007 - 2011 tables 1 and 2 show changes in cvd mortality rates according to individual - level baseline socioeconomic and demographic characteristics in the nlms . Education, income, and occupation were inversely associated with cvd mortality in both men and women, with individual - level socioeconomic patterns being similar during 1979 - 1989 and 1990 - 1998 . Although cvd mortality rates for almost all socioeconomic and marital status groups, especially for men, declined significantly between the two time periods, the social gradients in mortality persisted . Men and women with low education and incomes had 46 - 76% higher cvd mortality risks than their counterparts with high education and income levels . Men in clerical, service, farming, craft, repair, construction, and transport occupations, and manual laborers had 30 - 58% higher cvd mortality risks than those employed in executive and managerial occupations . Among women, those employed in sales and service occupations, respectively, had 17% and 29% higher cvd mortality risks, and those in transport occupations had 2.6 times higher mortality risks in 1990 - 1998 than those in executive and managerial occupations . Unemployed men but not women were significantly more likely to die from cvd than their employed counterparts . Both men and women with disabilities had 2.1 - 2.6 times higher risks of cvd mortality than their employed counterparts . In 1990 - 1998, self - employed men had the lowest cvd mortality risk, whereas women working in the government sector had the lowest mortality risk . Divorced / separated, never married, and widowed individuals had 15 - 32% higher cvd mortality risks than their married counterparts . Age - adjusted death rates per 100,000 person - years of exposure and relative risks of cardiovascular disease (cvd) mortality among us males aged 25 + years: the national longitudinal mortaliy study, 1979 - 1998 age - adjusted death rates per 100,000 person - years of exposure and relative risks of cardiovascular disease (cvd) mortality among us females aged 25 + years: the national longitudinal mortaliy study, 1979 - 1998 figure 1 shows changes in relative contributions of cvd and cancer, the top two leading causes of death, to overall us mortality over time . Even though cvd has become relatively less important over time as a cause of death, it still accounted for 31% of all us deaths in 2013 . About 23% of all deaths in 2013 were due to cancer . In 1969, on the other hand, cvd accounted for 53% of all us deaths, and there were 3 times as many deaths from cvd as from cancer . In terms of age - specific risks, cvd is a more prominent cause of death than cancer for infants and at older ages 75 years and beyond, while cvd and cancer exert similar mortality risks between ages 20 and 74 years (figure 1). Relative contribution of cardiovascular disease (cvd) and cancer to overall mortality and age - specific patterns in cvd and cancer mortality, united states, 1969 - 2013 figure 2 presents annual cvd mortality trends from 1969 to 2013 for the total us population and by race / ethnicity and sex . During 1969 - 2013, mortality rates declined consistently between 1990 and 2013 for american indians / alaska natives (aians), asians / pacific islanders (apis), and hispanics at average annual rates of 2.79%, 3.11%, and 2.47%, respectively . Trends in cardiovascular disease (cvd) mortality by race / ethnicity and sex, united states, 1969 - 2013 while absolute black / white differences in cvd mortality, as measured by rate differences, narrowed somewhat during the last two decades, racial disparities in cvd mortality, as measured by rate ratios, widened consistently between 1969 and 2013 (figure 2). Compared to whites, blacks had 12% higher cvd mortality in 1969 but 38% higher mortality in 2007 . In 2013, blacks had 30% higher cvd mortality than whites, but apis, aians, and hispanics 25 - 39% had lower cvd mortality than whites . Area socioeconomic gradients (rate ratios) in cvd mortality increased substantially during 1969 - 2011 (figure 3). Compared to those in the most - affluent group, individuals in the most - deprived area group had 11% higher cvd mortality in 1969, but 40% higher mortality in 2007 - 2011 . During 1969 - 2011, even though cvd mortality rates decreased by at least 62% for all deprivation groups, more deprived groups had higher mortality rates than less deprived groups in each year / time - period (figure 3). Cvd mortality declined at a much faster pace among individuals in the most affluent group than those in more deprived groups . During 1969 - 2011, cvd mortality in the five most - to - least deprived groups decreased at average annual rates of 2.20%, 2.48%, 2.54%, 2.66%, and 2.71%, respectively . Absolute socioeconomic disparities in cvd mortality for the total, male, and female populations, as measured by rate differences between the most and least deprived quintiles, also widened between 1982 and 2012 . Us cardiovascular disease (cvd) mortality by area socioeconomic deprivation index, 1969 - 2011 cvd mortality rates decreased with decreasing deprivation levels for both white and black populations, with socioeconomic gradients in cvd mortality increasing consistently during 1969 - 2011 for both racial groups (data not shown for brevity). Absolute socioeconomic inequalities also widened over time for both whites and blacks . During 2007 - 2011, cvd mortality rates varied from a low of 118.23 deaths per 100,000 population for aians in the least - deprived group to a high of 334.59 for blacks in the most - deprived group . Whites, blacks, aians, apis, and hispanics had, respectively, 1.36, 1.32, 1.76, 1.27, and 1.42 times higher cvd mortality in the most deprived group than in the least deprived group (figure 4). Cardiovascular disease (cvd) mortality by race / ethnicity and socioeconomic deprivation level, united states, 2007 - 2011 tables 1 and 2 show changes in cvd mortality rates according to individual - level baseline socioeconomic and demographic characteristics in the nlms . Education, income, and occupation were inversely associated with cvd mortality in both men and women, with individual - level socioeconomic patterns being similar during 1979 - 1989 and 1990 - 1998 . Although cvd mortality rates for almost all socioeconomic and marital status groups, especially for men, declined significantly between the two time periods, the social gradients in mortality persisted . Men and women with low education and incomes had 46 - 76% higher cvd mortality risks than their counterparts with high education and income levels . Men in clerical, service, farming, craft, repair, construction, and transport occupations, and manual laborers had 30 - 58% higher cvd mortality risks than those employed in executive and managerial occupations . Among women, those employed in sales and service occupations, respectively, had 17% and 29% higher cvd mortality risks, and those in transport occupations had 2.6 times higher mortality risks in 1990 - 1998 than those in executive and managerial occupations . Unemployed men but not women were significantly more likely to die from cvd than their employed counterparts . Both men and women with disabilities had 2.1 - 2.6 times higher risks of cvd mortality than their employed counterparts . In 1990 - 1998, self - employed men had the lowest cvd mortality risk, whereas women working in the government sector had the lowest mortality risk . Divorced / separated, never married, and widowed individuals had 15 - 32% higher cvd mortality risks than their married counterparts . Age - adjusted death rates per 100,000 person - years of exposure and relative risks of cardiovascular disease (cvd) mortality among us males aged 25 + years: the national longitudinal mortaliy study, 1979 - 1998 age - adjusted death rates per 100,000 person - years of exposure and relative risks of cardiovascular disease (cvd) mortality among us females aged 25 + years: the national longitudinal mortaliy study, 1979 - 1998 cardiovascular disease mortality rates have decreased substantially for all socioeconomic and racial / ethnic groups in the united states . Despite the impressive overall decline in mortality, socioeconomic and racial disparities in cvd mortality have remained marked and increased over time, as relative inequalities in mortality widened consistently between 1969 and 2013 . During 1969 - 2013, whites and those in more affluent groups experienced faster declines in cvd mortality than blacks and those in more deprived groups respectively, which contributed to the widening gap in cvd mortality . Absolute inequalities in cvd mortality, as measured by mortality rate differences, also widened between black and white populations and across deprivation groups during the past three decades . These marked and growing social inequalities in cvd mortality runs counter to the goals of the national health initiative that calls for further reductions or elimination of health inequalities in the united states, including those in cardiovascular disease, by 2020 . Area - based socioeconomic differences in cvd mortality reported here are consistent with those at the individual level . Cardiovascular disease mortality rates in 1979 - 1989 and 1990 - 1998 by individual - level socioeconomic and demographic characteristics presented herein should serve as the baseline data for comparison with more recent mortality follow - up data in the nlms and for comparison with international patterns . Socioeconomic and racial / ethnic disparities in cvd mortality reflect inequalities in the social environment (e.g., material living conditions, social support, and stress), behavioral risk factors such as smoking, obesity, physical inactivity, disease prevalence, and healthcare access and treatment . In the united states, racial / ethnic minorities such as blacks, american indians / alaska natives, and hispanics they have significantly lower education and income levels and higher rates of poverty, unemployment, and lack of health insurance than whites. [1,23 - 25] during the past 4 decades, poverty rates for blacks and hispanics have been 2 - 3 times higher than the rate for whites, and the unemployment rate for backs has consistently been twice that for whites . The substantial white - black gap in median family income has persisted, and absolute racial differences in income have widened over time . In general, ethnic minorities and individuals with lower ses or in more deprived areas of the united states have reduced access to health care and are more likely to forego or delay preventive or needed medical care than their more advantaged counterparts . With reduced access to preventive care and early detection services, individuals in disadvantaged minority groups are more likely to be diagnosed with advanced disease and have lower survival rates even after controlling for socioeconomic characteristics . Racial / ethnic and socioeconomic patterns in risk factors such as smoking, hypertension, and serum cholesterol are generally consistent with those in cvd mortality . Smoking prevalence is higher in american indians / alaska natives, whites, and blacks than asians . Smoking prevalence is also substantially higher in lower ses groups or more deprived areas, and has fallen much more rapidly in higher ses groups than in more disadvantaged groups . In 2012, the prevalence of hypertension was 38% higher in blacks than whites, whereas those with low education and family income levels had an approximately 50% higher prevalence of hypertension than their low - ses counterparts . There is a consistent income gradient in high cholesterol levels, with americans at lower income levels having moderately higher serum cholesterols than their higher - income counterparts . Obesity prevalence is higher among lower socioeconomic groups and among blacks, american indians / alaska natives, and hispanics . Social class gradients and racial disparities in us obesity rates have persisted for the past 4 decades . With obesity and diabetes prevalence continuing to rise in the united states and smoking and physical inactivity levels being relatively high among several racial / ethnic and sociodemographic groups, further declines in us cardiovascular disease mortality are not a given . Indeed, with prevalence of many chronic disease risk factors on the rise in many parts of the world, the global burden of cardiovascular disease and other non - communicable diseases such as cancer and diabetes is expected to increase further, particularly in low- and middle - income countries, where over 80% of all cvd deaths occur . The temporal patterns in cvd mortality shown here are consistent with those observed previously for the united states, where socioeconomic disparities (in terms of relative inequalities) increased over time even as overall cvd mortality rates declined markedly . A previous nlms analysis based on the 1979 - 1989 follow - up showed substantial inverse socioeconomic gradients in cvd mortality, with men and women with low levels of education and family income having 35 - 63% higher adjusted risks of cvd mortality than those with high education and income levels, a finding compatible with our study . Also consistent with our study, the previous nlms study found 32 - 45% lower cvd mortality risks in hispanic women and men than their non - hispanic white counterparts, even after adjusting for socioeconomic and demographic characteristics . Increasing socioeconomic disparities in cvd mortality shown here are consistent with those observed for several european countries although the magnitude of ses disparities in the us tends to be greater than that for some countries in southern europe. [29 - 33] like the united states, social class gradients in cardiovascular disease and ischemic heart disease mortality in the united kingdom and other european countries increased as mortality rates declined more rapidly for those in lower social class groups or more deprived areas. [29 - 33] reductions in cvd mortality rates for all socioeconomic and racial / ethnic groups in the us have contributed substantially to gains in life expectancy over time and must be seen as a major public health achievement . However, the growing socioeconomic and racial disparities in cvd mortality remain a major public policy concern . Because cardiovascular diseases are the leading cause of death and account for nearly one - third of all us deaths, these widening inequalities in cvd mortality contribute greatly to overall levels of health and mortality inequalities in the united states . These disparities in mortality reflect substantial and continuing gaps in cvd prevention and control efforts across various population subgroups . Higher inequality in social and behavioral risk factors is perhaps an important contributing factor for the united states unfavorable standing in cvd mortality relative to many other organization for economic cooperation and development (oecd) countries. [32 - 34] for example, canada, united kingdom, australia, germany, sweden, norway, denmark, and switzerland have significantly lower mortality rates from ischemic heart disease, and the rates for south korea, japan, and france are 60 - 70% lower than those for the united states . While changes in the social patterning of behavioral risk factors such as smoking, diet, physical inactivity, and alcohol use may have been largely responsible for temporal social inequalities in cvd mortality, the inequalities in lifestyle factors are themselves largely determined by underlying social and economic factors . Behavioral and health care interventions would likely be less effective without addressing the larger societal / social and economic forces that give rise to inequalities in behavioral risk factors in the first place . A broad course of policy action with a clear emphasis on the wider social determinants is, therefore, needed to tackle the problem of growing inequalities in cvd mortality . Health and social policy interventions such as improved access to health services and reductions in inequalities in education, poverty, unemployment, occupation, and housing are essential for tackling long - term health inequalities in cvd mortality between socioeconomic and racial groups in the united states . Black / white disparities in mortality from cardiovascular disease (the leading cause of death in the us for the past eight decades) rose consistently betzeen 1969 and 2013.lower levels of area- and individual - level socioeconomic position were associated with substantially higher rates of cardiovascular - disease mortality among us men and women during the study period.area socioeconomic inequalities in cardiovascular - disease mortality increased consistently between 1969 and 2011 for the total us population, whites, and blacks as higher socioeconomic groups experienced faster mortality declines . Absolute inequalities in cardiovascular - disease mortality also widened over time.increasing social inequalities in us cardiovascular - disease mortality may be related to increasing temporal differences in material living conditions and behavioral risk factors such as smoking, obesity, physical inactivity, and unhealthy diet between social groups and geographic areas.from a policy standpoint, narrowing the socioeconomic gap between affluent and disadvantaged areas has the potential to reduce cvd and hence overall mortality . With prevalence of many chronic disease risk factors on the rise in the developing world, the global burden of cardiovascular diseases and other non - communicable diseases such as cancer and diabetes is expected to increase further, particularly in low- and middle - income countries where over 80% of all cvd deaths occur . Black / white disparities in mortality from cardiovascular disease (the leading cause of death in the us for the past eight decades) rose consistently betzeen 1969 and 2013 . Lower levels of area- and individual - level socioeconomic position were associated with substantially higher rates of cardiovascular - disease mortality among us men and women during the study period . Area socioeconomic inequalities in cardiovascular - disease mortality increased consistently between 1969 and 2011 for the total us population, whites, and blacks as higher socioeconomic groups experienced faster mortality declines . Absolute inequalities in cardiovascular - disease mortality also widened over time . Increasing social inequalities in us cardiovascular - disease mortality may be related to increasing temporal differences in material living conditions and behavioral risk factors such as smoking, obesity, physical inactivity, and unhealthy diet between social groups and geographic areas . From a policy standpoint, narrowing the socioeconomic gap between affluent and disadvantaged areas has the potential to reduce cvd and hence overall mortality . With prevalence of many chronic disease risk factors on the rise in the developing world, the global burden of cardiovascular diseases and other non - communicable diseases such as cancer and diabetes is expected to increase further, particularly in low- and middle - income countries where over 80% of all cvd deaths occur.
The geographic distribution of very low fertility among the countries of the world, especially within europe, remains a puzzle . Rapidly falling fertility in these western countries, many scholars argued, could be explained by the massive entry of women into the extra - domestic labor force . Others, doubting that a purely economic approach would prove adequate, identified basic culture change as generating very low fertility . Along these lines, second demographic transition (sdt) theorists pointed to a shift in cultural values linked both to a move away from familism toward self - realization and a shift from religious attachments toward secularism . The fact that by the early 1990s italy and spain emerged as the countries with the lowest fertility proved a major surprise, if not an embarrassment . In the european context, both countries showed unusually low rates of female labor force participation (flfp) and relatively strong family bonds and religious institutions . As chesnais (1998, p. 91) pointed out, no official population forecast, either national or international, had anticipated a total fertility rate of 1.2 for any country, [much] less for mediterranean countries, which are still commonly viewed as laggards and...family - oriented . This outcome is probably the biggest surprise of european demographics at the end of the present century . The longstanding fertility differential between northern and southern europe was unexpectedly reversed . Countries viewed as traditional, catholic, and family - oriented inexplicably had markedly lower fertility than those that were protestant, more secular, and had weaker family ties (chesnais 1996, p. 729). In this article we provide a detailed empirical analysis of the italian case to shed light on the adequacy of current demographic theorizing on very low fertility . We begin by introducing some major threads in the arguments and offer relevant background on italy . We then introduce our data and present a series of event history models with longitudinal microdata . We conclude by considering the theoretical lessons of the italian case . To date, economic theories of various kinds have dominated the theoretical debate on very low fertility . Following becker (1976) and other neoclassical economists (mincer 1963), much work has focused on increased female autonomy, the movement of more women into the labor force, and calculations of the direct and indirect costs of childbearing to the family economy . At a more macro - level, easterlin (1976) and colleagues (easterlin and cummins 1991) have advanced a theory of relative economic deprivation, linking fertility decisions to economic opportunities related to relative cohort sizes and to judgments based on expected levels of economic well - being linked to demographic and economic cycles . As noted by macunovich (1997), neither theory seemed to be born out by the course of fertility in the world s wealthier countries in recent years . The economic feature that has most attracted theorists attention has been increasing levels of flfp . Economic theory successfully predicts the substitution of activity away from childbearing and childrearing with growing wage opportunity for women at the individual level . Yet at the country level the correlation between flfp and tfr reversed from being negative in the 1960s to sharply positive in the 1990s (kgel 2004; morgan 2003). Bernhardt (1993, p. 32) has argued that the inhibiting effect of work on fertility has been at least partially removed with the help of social and institutional arrangements, citing in particular the provision of publicly funded childcare, maternity leave, and tax benefits (gauthier 1996). In the past several years, researchers have turned attention to various social policies and public institutions that might affect the relationship between women s work and the cost of childbearing . The reasoning suggests that the differences in both labor market participation and fertility rates in different western countries can largely be attributed to the characteristics of institutions like childcare system, parental leave arrangements, and labor market flexibility (engelhardt and prskawetz 2004; rindfuss et al . In particular, it has been noted how in scandinavian countries and france, public policies encourage both female work and childbearing by easing the childcare burden for dual earner families . This is done through public support in the form of affordable childcare services, generous maternity leaves, and protection of part - time employment . In anglo - saxon countries the flexible frame for part - time employment and the private childcare services offered by the market may compensate for more limited welfare provisions . By contrast, it is argued, in southern europe labor market rigidities and imperfections biased toward full - time or limited employment contracts, an inadequate supply of public childcare, and other institutional constraints, combine to make work and parenthood highly incompatible (del boca and pasqua 2005b). It is further argued that while relaxation of these labor market rigidities (to enable more part - time employment) may increase overall female labor force participation, the shift may come with the cost of lower fertility in these settings of southern europe (del boca et al . The incompatibility impasse in southern european countries seems to explain the survival of the negative relationship between flfp and fertility in these settings (kgel 2004). Trends in the variables that would be representative for the role incompatibility hypothesis and the ease in combining work and child - rearing cannot be related to the trends in fertility . There is significant european cross - national variation in the use of public and private childcare, but little evident correlation with fertility (kiernan 1998). Taking a different perspective, a number of scholars have argued that no adequate theory of fertility can be developed that does not incorporate an understanding of culture (kertzer 1995, 1997). Most prominent in this regard has been second demographic transition (sdt) theory (van de kaa 1987; lesthaeghe and surkyn 1998, p. 8). This approach identifies a shift toward individualization and secularization and links it to the almost contemporaneous fall of fertility to below replacement . Van de kaa and other second transition theorists attribute this transition to a basic shift toward values emphasizing the rights and self - fulfillment of individuals (van de kaa 1987, p. 5) and away from traditional family - oriented values, citing the dramatic increase in late - twentieth - century europe in divorce, cohabitation, and non - marital childbearing . Yet the countries in europe that currently have the lowest fertility are those showing the strongest commitment to the traditional forms of family formation, with relatively low divorce, non - marital cohabitation, and illegitimacy rates, and in the propensity of adult children to remain in their parental household until marriage and to live very close to them thereafter (mcdonald 2001). To gain better insight into these different mechanisms secular change, labor force participation, public institutions, and family culture it is desirable to go beyond aggregate inter - country comparisons and to look more carefully at changing fertility behavior within a country . Italy s well - known position in cross - national comparisons of fertility trends does not tell much about the historical variation in the path of fertility decline across its regions . While it is true that fertility declined in all regions, such decline started at very different moments and proceeded at very different speeds in the various regions . In tracing the geographical pattern of marital fertility decline across italian provinces (a lower level of aggregation than the regions) through the 19th and the 20th centuries, livi bacci observes that in the areas with an early decline in fertility [the north], neo - malthusian attitudes had spread at an uneven pace because of deep economic and social differences among the various sectors of the population . [...] on the other hand in the south, where the population is perhaps more homogeneous in its socio - economic characteristics and certainly culturally less differentiated, acceptance of neo - malthusian attitudes was relatively slow and late and did not generate pronounced fertility differentials replacement levels of fertility were reached in parts of the north beginning with the 1910 birth cohort, while as late as the early 1980s the tfr in a number of southern regions still stood above replacement.1 over the last two decades of the 20th century, fertility in northern and central italy remained stable, while the southern regions experienced continuing sharp fertility decline . By the century s end, campania (centered on naples), although still having the highest fertility in the country, had a tfr of only 1.5 . Remarkably, sardinia, known for its economic underdevelopment, rugged terrain, and traditionalism, and which in 1960 had the highest tfr in italy (3.5), as might comfortably comport with both economic and sdt theory, had italy s lowest fertility (1.04) by the end of the century (dalla zuanna and crisafulli 2002, table 2). Despite the current observed convergence of regional fertility rates, italian regions are still marked by important variations in demographic, economic, and social patterns . This regional and temporal variation makes the italian case an especially fruitful one for theoretical exploration of the impact of economic factors and cultural values on fertility . Substantial variation also characterizes the levels of female labor force participation (flfp) in the different regions, yet the correlation between regional 2003 tfr and regional flfp (of women 2534) is a modest 0.24 . Women in the north of italy are much more likely to have paid employment . In the mid-1990s, 64% of women aged 2049 in the northwest, but only 36% in the south did paid work . Even more strikingly, 41% of the southern women had never been in the labor force, compared to only 7% of those in the northwest (bernardi 1999, p. 753). Indeed, the increase in flfp has been quite modest in the south, with the proportion of women who had ever entered the labor force rising only from 41% among those born before 1929 to 51% in the 194458 birth cohort (compared to 84% in the north) and little sign of any increase since then (barbagli et al . 2003, table 1.8). Among women with children under age six in the mid-1990s, 62% of the northerners and only 31% of the southerners were employed (sabbadini 1999, table 3.5). A 1998 national sample survey (famiglia e soggetti sociali, fss) asked all individuals if they had ever been in the paid labor force . The persistence of strong regional differences, particularly a north - south contrast, is clear . In the south, 50% of the 194150 birth cohorts had never entered the labor force, while in the north only 20% had refrained from participation . By the 196170 cohorts the figure had increased to 54% in the south, while it had decreased to around 10% in the northern regions . A third element of regional variation is represented by the ideational dimension related to gender norms and union formation . There is consistent empirical evidence that egalitarian gender norms and spousal (female) autonomy are stronger in the north (sabbadini 1999, table 6.3), where premarital cohabitation rates and divorce rates are also notably higher (1999: table 4.7; barbagli 1990). Behavioral and attitudinal indicators that tap individualization vary regionally, declining as one moves from the most secularized northwest to the traditional south (table 1). By contrast, there is one cultural feature that shows relatively limited regional difference and that is the propensity of young adults to live with their parents before marriage (sabbadini 1999, table 5.3) and to choose residential arrangements very close to their parents thereafter (cioni 1997; bordignon et al . Table 1descriptive statistics for ilfi samplevariablesmean values1st union1st birth2nd birthindividual traits (tv = time - varying)northwest (tv) [ref]0.120.150.14northeast (tv)0.250.300.26center (tv)0.270.280.28south (tv)0.360.270.32cohort 194150 [ref]0.310.340.37cohort 1951600.330.360.36cohort 1961700.360.300.27mother worked0.360.340.34father s education elementary0.660.700.72father s education middle 0.130.130.12father s education high school [ref]0.100.090.08father s education post - secondary0.110.080.08father died (tv)0.050.170.16mother died (tv)0.020.080.06education of respondent secondary or lessna0.560.59education of respondent high school [ref]0.340.33education of respondent post - secondary na0.100.08employed (tv)na0.560.52 in a civil union (religious ref)0.070.05entered union, age 15190.16entered union, age 20240.51entered union, age 2529 [ref]0.27entered union, age 30340.05entered union, age 35390.01entered union, age 40 + 0.00age at first birth, 15190.09age at first birth, 20240.42age at first birth, 25290.34age at first birth, 3034 [ref]0.12age at first birth, 35490.04n women (at spell onset)3,4002,9161,914n women (at spell onset)3,4002,9161,914na = few women have completed education or been employed at the onset of risk (age 15) for union; subspells incorporate changes to these variables . Time varying covariates are lagged 12 months descriptive statistics for ilfi sample na = few women have completed education or been employed at the onset of risk (age 15) for union; subspells incorporate changes to these variables . Time varying covariates are lagged 12 months while a regional comparison leads to doubts about the adequacy of an explanation focusing on women s work, attempts to link regional level cultural differences of the sort theorized by sdt theorists to fertility levels lend little support to this alternative . Of all marriages in italy in 2001, over a quarter were conducted civilly, generally in the local town hall . In italy s largest northern regions piedmont, lombardy, veneto, liguria the proportion of religious marriages ranged from 60 to 69% . In emilia - romagna and tuscany, in the heart of italy s red belt, with its anticlerical and left - wing tradition, by contrast, in no part of the mainland south was the percentage of religious marriages under 80%, and in the deep south (puglia, basilicata, calabria) it was near 90% . Yet plotting regional tfr against regional percentage of religious marriages in 2003 offers little evidence of any link between secularization and low fertility (figure not shown). Italy s least secularized region using this measure was the deep southern region of basilicata, where only 9% of marriages were celebrated outside a church, yet in 2003 basilicata s tfr was 1.20,2 actually below the national average . Regional - level analysis of the italian case fails to find much support for either flfp or sdt theories of very low fertility . We now turn to micro - level data to see whether the predicted relationships may, while obscured in regional level comparisons, be evident in individual behavior . We analyze data from the italian longitudinal family study (ilfi), the only ongoing prospective social survey in italy . Based on an original sample of 4,404 households within which all members are interviewed (9,770 individuals> 18 years old), ilfi includes detailed fertility histories that make it especially valuable for our analysis . The first wave of the panel took place in 1997 and we here analyze data through the first three waves (1997, 1999, 2001). The dynamic nature of the sample means that at every wave it loses all individuals who (a) died, (b) migrated abroad, or (c) became severely impaired; and it gains individuals who (a) reach age 18 and belong to the originally sampled households, and (b) enter via union or cohabitation . Ilfi is representative of the italian population nationally.3 looking at italian women born in 19411970 we examine three crucial life course transitions: marriage, the transition from marriage to first birth, and the transition from first to second birth . It is in these cohorts who began entering their reproductive years in 1955 and whose fertility primarily occurred between 1960 and 2001that the dramatic and puzzling fall of italian childbearing is most clearly manifest . Women in birth cohorts 19411970 were approximately 3060 years old at the time of the ilfi survey . For the youngest cohorts, we cannot observe all their childbearing exposure, although our models adjust for censoring . Premature mortality of selectively differential women could affect our results, although given limited female mortality at these ages it is likely that any such effect is slight . We analyze each of these three transitions with cox regression models and introduce a set of covariates designed to capture both fixed and time - varying traits of the women . Of course, it is only women who experience one of these cumulative life transitions who join the population of women at risk of experiencing the next transition . Thus, we can expect that successive transitions tap a pool of women who are increasingly selective . We present pooled and cohort - specific models, after having explored a large number of alternative specifications . Following our interest in weighing the potential value of theories that focus on women s work on the one hand, and secularization (sdt theory) on the other, we pay particular attention to two variables: (1) whether the woman is in the work force, which is lagged by one year so as to capture a possible causal effect; and (2) whether the woman was married in church or in a civil marriage . The number of cohabiting couples in the sample is too small to incorporate a separate measure of this form of union . We regard civil marriage as a compelling indicator of the secularization of italian society, consistent with the concept of the second demographic transition . Just because civil marriage is a strong measure of secularization, it has the limitation for our purposes of being relatively uncommon among the oldest cohorts . In the analysis to follow, we organize our presentation by event: first union, first birth, second birth . In each case among the factors often cited as contributing to very low fertility is a postponement of marriage to ever - later ages . Such a factor might be thought to be especially influential in italy, where childbearing remains closely tied to marriage . Italy has, in fact, been among the countries that have recently experienced a sharp rise in the age at first marriage . From 1985 to 2001 women s mean age at first marriage rose dramatically, from 23.8 to 28.5.4 in analyzing the transition to marriage we follow convention in taking age 15 to indicate the onset of risk and so model the duration from age 15 to (age at) first union . Women are censored at the time of the survey if they have not yet entered a union . In this analysis we take only formal unions (civil and religious marriages) to constitute an event ending the spell . Cohabitation is relatively rare in italy; thus, the sparseness of the events made cohabitation difficult to analyze separately . Furthermore our preliminary analysis suggested that cohabiters may be qualitatively different from those entering formal unions . In descriptive data via kaplan meier survival analysis, we observe dramatic differences by cohort, with the youngest cohort, born in 196170, showing a marked slowdown in marital timing while in both the 194150 and the 196170 cohorts about two - thirds of women had married by age 25, only 39% of women in the youngest cohort had done so by that age . In fact, our kaplan meier data indicate that after 20 years of exposure (accounting for censoring)that is at age 35about one - third of the youngest cohort had yet to enter a formal union.5 figure 1 presents the survival curves for the duration to first union, drawn separately by region . We define macroregion to be an aggregation from 20 administrative regions into 4 larger regions, as used in our introduction and by other analysts . Our entire ilfi sample born since 1940 (age 3060 at time of survey) is represented in these graphs . Here this lack of regional difference in marriage rates is itself noteworthy, in that, as we shall show below, our geographic depictions of fertility rates point to significant differences across the four macro regions of italy . 1duration to union by macroregion duration to union by macroregion we extend these descriptive portraits with regression models, in which we test for the relative influence of cohort, region, and a number of potentially influential traits of each woman . These include family background factors mother s work experience, father s education, death of mother and father as well as the woman s own education and employment status . Table 2 presents cox regression models for the prediction of duration from age 15 to first union . Coefficients are presented in relative risk format, and hence indicate the proportional shift in the baseline hazard due to a unit change in that variable, i.e. Membership in that category . Table 2cox regression models for union formation 2001 ilfi survey (all women 15 +) variablemodel 1model 2model 3model 4model 5pooled sample with controls for region and cohort onlypooled sample with standard controlscohort born 194150cohort born 195160cohort born 196170estimatestandard errorestimatestandard errorestimatestandard errorestimatestandard errorestimatestandard errorindividual traits (tv = time - varying)region (versus northwest)northeast (tv)1.0170.0740.9930.0720.8770.1001.1490.1490.9600.142center (tv)0.9250.0670.9570.0700.8600.0981.1070.1430.9570.140south0.9000.0640.8190.0600.6160.0700.8240.1101.1390.164cohort (versus 194150)cohort 1951601.0060.0531.128 0.060cohort 1961700.519*0.0290.6170.036mother worked0.8780.0420.9660.0800.9320.0740.7650.067father s education (versus diplome)father s education none - primary1.5130.1281.337#0.2011.290#0.1861.9120.276father s education secondary1.3350.1321.2050.2211.1260.1911.6510.272father s education post - diplome0.756 0.0830.8600.1640.627 * 0.1170.7870.158father died (tv)1.0090.0681.1810.1260.9580.1110.8520.117mother died (tv)0.9330.0931.0780.1560.7900.1450.9840.210respondent s education (versus diplome)education of respondent primary or less1.645*0.0881.5980.1571.6510.1451.6700.154education of respondent post - secondary1.3710.1251.473 0.2471.446 * 0.2221.2170.194employed (tv)0.827*0.0400.7010.0570.854#0.0710.9890.089n women2,5262,526788824914n observations10,91210,9123,1973,5084,207n events2,0392,039715725599log - likelihood14,573.32614,449.3224,183.1654,302.3243,728.474 * * p <0.001, * * p <0.01, * p <0.05, #p <0.10 cox regression models for union formation 2001 ilfi survey (all women 15 +) * * * p <0.001, * * p <0.01, * p <0.05, #p <0.10 model 1 includes only cohort and region . Women in the youngest birth cohort (196170) marry at about 52% of the rate of women in the reference (194150) cohort . Again there is no appreciable difference between the northwest macro region (reference)the area identified with the earliest attainment of very low fertility and the northeast and the center . Women in the south exhibit a relative risk of 90%, but this difference is not statistically significant.7 our next model introduces the standard set of covariates suitable for the prediction of first union . As mentioned above, these include measures of economic background, parental mortality, woman s own education, and, especially, employment . (death of mother, death of father, and woman s employment are all time - varying covariates .) In the presence of these additional personal traits the effects of region and cohort shift modestly . Regional effects become more pronounced (with all regions exhibiting lower relative risks compared to the northwest once controls are introduced). Women in the south are now shown to have about 20% lower marriage hazard rates (rrr = 0.818), and this is highly significant . The secular decline in propensity to marry remains, indicated by the significance of the dummy variable for the 196170 birth cohort . The increase in the magnitude of the effect of regional dummy variable for the south upon the inclusion of the employment measure is suggestive of a slightly different employment - marital regime in that region . Once we control for employment, women born in the south are actually even less likely to marry than women in other regions; their lower rate of employment partly masks this . Several other family background factors are strongly predictive of this first step in family formation . Women whose mothers worked when the women were aged 15 exhibit a marital hazard rate about 12% below those of other equivalent women . We find no effect of parental mortality on union formation.8 the education of the women is quite strongly predictive of the rate of entry into marriage, showing a u - shaped relationship . Women with low levels of education have a marriage rate 64% above women with moderate levels of education . On the other hand, women with high levels of education exhibit a hazard rate for duration to marriage 37% above the middle group.9 employment of the woman herself much contemporary theory about very low fertility, as discussed above, would suggest that employment would depress the family building activity of contemporary women . We ask not only whether this is true, but also the magnitude of the effect and the extent of its effect across time (birth cohorts) and across the successive family - building events we examine with ilfi . Model 2 of table 2 indicates that on balance women s employment operates to significantly and appreciably reduce the rate of entry into marriage to about 83% of otherwise equivalent women . This aggregate result (cohorts pooled) offers support for the view that the decline in fertility commences with lower rates of entry into marriage, in turn, partly driven by women s employment.10 models 35 split the cohorts and repeat the analysis; thus, these models allow the effects of traits to differ across the three cohorts of women . Women in the 194150 cohort have passed beyond the standard reproductive span by the time of the ilfi 2001 survey wave . Thus information for their union formation (as linked to fertility) and childbearing should be complete . For these women we find clearly visible effects of region, woman s own education, and employment . Women in the south make the transition to first marriage more slowly than women from other regions of italy . (women in the northeast and center show relative risk rates below unity, but these are not statistically significant .) At the same time we find that women whose education is low or high experience more rapid marriage the u - shaped effect . This statistical result may be worth particular consideration in this cohort, as the apparent inconsistency may point to competing mechanisms linked to some of the overarching processes we identified at the outset . Certainly aspiration for education and labor market participation are intertwined . As in the pooled results working women make the transition to marriage less quickly, about 30% below the transition rate of otherwise equivalent women . Some elements of this nuptiality process change by the time the next cohort, born 195160, is observed . In this case, regional differences are muted further, with almost no differential at all among northeast, northwest, and center . The point estimate for women from the south indicates a lower rate of transition into marriage, but this difference is not statistically significant . Personal and parental traits have some predictive value, in a direction mostly consistent with an effect in which higher socioeconomic status retards marriage . Women whose fathers had little education experienced more rapid rates of marriage, while children of highly educated fathers experience slower marriage . The woman s own education matters a good deal, and again, a u - shaped pattern is observed: poorly and highly educated women are quicker to marry . The employment effect is statistically significant, although its strength does not match that found in other cohorts . In this middle birth cohort, we observe that employed women enter unions at about 15% lower rates (rrr = 0.856) than other equivalent women . Our final cohort comparison turns to women born in 196170, who are aged 3141 at the time of the survey . We observe 914 such women and nearly two - thirds of them have married as of the survey date . (no coefficients are even marginally significant .) Yet personal background traits are predictive . Women whose fathers had little or no education (a decreasing fraction of the population of women over time) entered into marriage more quickly, while those whose mothers worked made this transition more slowly.11 the u - shaped effect of woman s own education is visible once again, although the only significant effect indicates that poorly educated women are quicker to marry . Perhaps most notable in the cox regression of this youngest cohort is the lack of any effect of the woman s own employment . It appears that with women s work becoming more common and, perhaps, normatively more accepted, it no longer has the effect on discouraging marriage that it formerly did . We turn now more directly to our individual - level fertility analysis with a look at the transition from marriage to first birth . Since cohabitation is relatively uncommon in italy, and since childbearing outside of a formal union is relatively rare (although increasing), we exclude cohabiting individuals from the risk set.12 in our sample, some 92% of all women who had entered a formal union experienced a birth by the 2001 wave of the ilfi survey . As was to be expected, successive cohorts show a slowing down of transition to first birth . While nearly 90% of the 194150 cohort experienced a birth within 5 years of marriage, only about 80% of the 196170 cohort did so . But unlike the pattern we found in transition to first marriage, the transition from marriage to first birth showed clear regional differences across all cohorts (fig . 2). Women in the south proceeded more quickly to a first birth after marriage than did women elsewhere; 92% of women in the south had a child within five years of marriage compared to 83% of women in the northwest . 2duration to first birth by macroregion duration to first birth by macroregion a simple cox regression model with regional and cohort covariates alone confirms these graphical differences . Table 3 model 1 points to statistically significant and high rates of childbearing in the center and south, with progressively slower rates of the transition to parenthood across cohorts . This model includes covariates used above for union formation, but, to provide a test of sdt, adds a dummy variable for type of union: civil marriage (versus religious). Economic theory argues that woman s employment (a time - varying covariate) should serve to decrease the rate of transition to first birth . Education and socioeconomic background should work in corresponding ways, with higher status women slower to make the transition . A strict interpretation would suggest that regional (and cohort) differences should be reduced or even eliminated on the basis of these additional controls . Persistent effects not consistent with the standard explanation would suggest that regional differences and marital union type differences would remain, even in this more comprehensive model . Table 3cox regression models for first birth 2001 ilfi survey (all women in a formal union)variablemodel 1model 2model 3model 4model 5pooled sample with controls for region and cohort onlypooled sample with standard controlscohort born 194150cohort born 195160cohort born 196170estimatestandard errorestimatestandard errorestimatestandard errorestimatestandard errorestimatestandard errorindividual traits (tv = time - varying)region (versus northwest)northeast1.0290.0801.0130.0791.1410.1350.8650.1191.0520.180center1.208 * 0.0931.204 * 0.0931.214#0.1421.0680.1471.3130.224south1.583*0.1201.4470.1141.4090.1681.293#0.1861.6980.287cohort (versus 194150)cohort 1951600.8560.0470.8620.049cohort 1961700.6960.0410.7160.044mother worked1.0430.0520.9240.0791.1440.0961.1320.108father s education (versus diplome)father s education none1.0360.0940.9240.1451.1760.1880.9650.156father s education low1.0180.1090.8930.1721.1790.2180.9500.177father s education high0.9310.1101.0970.2161.0530.2100.5970.142father died (tv)1.0190.0671.0300.1070.9410.1101.1060.144mother died (tv)0.9690.0970.8480.1231.0800.1910.9930.225respondent s education (versus diplome)education of respondent low1.0120.0590.9900.1030.9720.0961.0500.107education of respondent high1.1030.1051.0290.1791.0480.1601.2930.234employed (tv)0.813*0.0420.824 0.0700.839 * 0.0740.7380.074 in a civil union (versus religious)0.7280.0801.1300.2810.673 * 0.1070.606 * 0.126union formation versus age 2529entered union, age 15191.494*0.1201.2100.1631.6920.2301.7230.275entered union, age 20241.2990.0771.3960.1361.275 * 0.1391.1870.128entered union, age 30340.9050.1111.2080.2460.7820.1630.7570.182entered union, age 35390.290*0.0930.2160.0990.4590.2361.1811.200entered union, age 40 + 1.6701.2011.9711.450n women1,9861,986700703583n observations2,7772,777958993826n events1,8321,832674657501log - likelihood12,386.09212,325.4933,814.3283,741.4302,764.664 * * * p <0.001, * * p <0.01, * p <0.05, #p <0.10 cox regression models for first birth 2001 ilfi survey (all women in a formal union) * * * p <0.001, * * p <0.01, * p <0.05, #p <0.10 in table 3, model 2 we find only modest effects of some of the socioeconomic background factors; mother s work, father s education, and parental mortality are all non - significant in this model . Even the woman s own education offers little additional power to predict first birth transition . In this model regional and cohort effects are reduced only slightly in magnitude from model 1 . Thus women in the center and south make the transition from marriage to first birth at higher rates, while women in recent cohorts make the transition more slowly, even after adjusting for other characteristics . We also include in this model covariates for woman s age at marriage . Collectively these five dummy variables (with marriage at age 2529 as the reference) are highly significant . We observe that women who marry at the youngest ages make the transition to first child more rapidly, all else equal . Women who marry at ages 3539 make this transition much more slowly than the women in the 2534 cohorts.13 there is no evidence of hastening the childbearing transition for women who enter (first) marriage at a later age . Importantly, our primary measures of both flfp theories and sdt theory regarding very low fertility woman s employment and marital union type are strong predictors of the progression from union to first birth . The relative risk of first birth transition for employed women is 0.81 and is one of the most powerful predictors in the model . A similar effect (0.73) this effect is highly significant . For a woman who is both employed and in a civil union together indicators of departure from a more traditional family building style the relative risk ratio is 0.59 (rrr = 0.813 * 0.728). In other words, for such women their rate of transition from marriage to first birth is 59% that of otherwise equivalent women . We extend the analysis to look at this transition separately by cohort, and estimated coefficients appear in models 35 of table 3 . In the 194150 cohort we find that women in the center and south experienced higher rates of first birth transition . In the complete model, almost no covariates for background socioeconomic traits are statistically significant . This older cohort had few cases of civil marriage and so it may not be surprising that no significant differences between women married civilly and religiously can be found here . We still find a strong effect of employment (rrr = 0.824), not much different from the model which pools across all three birth cohorts . It is quite clear that employment, even in these relatively early cohorts, is strongly and negatively associated with the transition to first birth.14 coefficients for age at union are not particularly consistent or stable . Still there is evidence that women who marry at younger ages (1524) make the first birth transition more rapidly . Again the background traits of parental characteristics and woman s own education do little to improve prediction in the model . While women in the south and central macro regions exhibit somewhat higher rates of transition, the associated coefficients do not achieve statistical significance . (there are 703 women at risk in this cohort .) By contrast, effects for employment and for union status are strong and highly significant . Employed women have a relative risk of first birth about 16% below other equivalent women in their cohort . Women in a civil union have a 33% lower relative risk . As for the pooled sample these two traits in combination (employed women in a civil union) predict a much lower rate of entry into childbearing . Here again, women who marry at an early age (1524) display a more rapid rate of making the transition to first birth . The story for the 196170 cohort differs in several important respects from the older two cohorts . These women are about age 3141 in 2001 at the time of the last ifli wave we use here . They thus have not completed their reproductive span, so that any additional timing shifts anticipated after 2001 cannot be captured by our data . Notably women in the south (rrr = 1.70; p <women in the center also progress more rapidly, although this difference is only significant at the 10% level . Parental background traits are again of modest predictive value, although we observe that women with fathers of the highest level of education have lower childbearing rates (rrr = 0.597; p = 0.03). In this youngest cohort of 583 women, what does show through is that women who marry as teens have much higher transition rates than those who marry in their twenties and early thirties . Here, though, the impact of bridal pregnancy undoubtedly weighs heavily . Being employed and in a civil (versus religious) union once again exert strong downward effects on predicted fertility . Employed women exhibit hazard rates some 30% below non - working women . As was the case with previous cohorts, the combination of working and being in a civil union serves to lower the predicted rate of transition from union to first birth appreciably, here by over one - half . We also investigated alternative models for the transition to first birth . In parallel to our examination in the case of union formation, we tested for the effect of part - time versus full - time employment . In a test of constraints across coefficients, we accept the null hypothesis of no difference in the effect of full- and part - time employment . Also, we tested for the influence of the number of siblings of the woman . A greater number of siblings might be expected to proxy a family background in which traditional roles and/or larger family sizes are valued . (in turn, this expectation would lead to a prediction of first birth occurring more rapidly .) We find no evidence of this, as the covariate for number of siblings provided no additional predictive power beyond the characteristics already in the model . We also examined an alternative model that included interaction terms between woman s own education and age at union . These are added to both the pooled and cohort - specific models, in which the first - order covariates for education and age at union appear as in table 3 . The joint test of the group of covariates indicates that they are not collectively significant . This indicates that there is no identifiable further alteration of the effect of education across women who enter unions at differing ages . The third and final element of our event history analysis focuses on second birth . In our ilfi sample of women in the cohorts born between 1941 and 1970, some 1,914 have a first birth, and of this group, 72% go on to bear a second child within the observation period . Not only is this a (necessarily) smaller sample than the number of women at risk of first union and first birth, it is more selective as well . There are no pronounced cohort differences in the transition to second birth, although it is evident that the 194150 cohort made the transition from first to second birth more rapidly than succeeding cohorts . Tabulations of the survivor function point to 5-year transition fractions of 59%, 51% and 53% for the cohorts successively . The youngest cohort is slow to make the transition for about 4 years following first birth, but after that point makes the transition more rapidly so that it reaches a crossover with the other two cohorts within a decade of exposure . It is worth recalling that the underlying sample contains more women in each of the older two cohorts than in the youngest, since the youngest cohort is least likely to marry and have a first birth, requisites for being in the risk set for second birth . 3 shows no discernible differences across northeast, northwest, and central for the five years following first birth, women in the south make this transition much more rapidly . 3duration to second birth by macroregion duration to second birth by macroregion table 4, model 1 points to statistically significant and somewhat higher rates of childbearing in the northeast and the center, compared to the northwest . But strikingly, the relative risk for the south is nearly double that for these other two regions and close to three times the rate found in the northwest . Cohort effects are present, with both the 195160 and 196170 cohorts displaying a hazard rate about 15% below that of the reference group of women in the 194150 cohort . The fact that the 195160 cohort differs little from the 196170 cohort in second - birth transition while it does differ in first birth transition (see above) suggests that restriction of fertility at higher parities (even 2nd) had begun to make its way into family building practice in this middle cohort, who were themselves passing through key years of the reproductive span in the 1970s and 1980s . Table 4cox regression models for second birth 2001 ilfi survey (all women with a first birth and in a union)variablemodel 1model 2model 3model 4model 5pooled sample with controls for region and cohort onlypooled sample with standard controlscohort born 194150cohort born 195160cohort born 196170estimatestandard errorestimatestandard errorestimatestandard errorestimatestandard errorestimatestandard errorindividual traits (tv = time - varying)region (versus northwest)northeast1.199#0.1221.211#0.1241.1660.1691.0440.1891.866 * 0.515center1.211#0.1211.229 * 0.1251.0930.1571.0860.1982.1920.590south2.7210.2622.5610.2522.4840.3382.4920.4483.7270.986cohort (versus 194150)cohort 1951600.8430.0540.8190.053cohort 1961700.832 0.0600.797*0.058mother worked1.130 0.0661.0831.0521.211#0.1181.211#0.141father s education (versus diplome)father s education none0.8840.0990.640 * 0.1160.9980.1931.0890.240father s education low0.8690.1130.587 * 0.1280.9630.2141.1040.276father s education high0.774#0.1110.573 * 0.1280.8330.2120.9960.295father died (tv)0.9440.0670.9800.1080.8530.1051.0070.156mother died (tv)0.9040.0970.9080.1400.9370.1830.9680.243respondent s education (versus diplome)education of respondent low1.0160.0701.0150.1201.0180.1170.9510.123education of respondent high1.1260.1250.648 * 0.1391.3110.2331.653 * 0.333employed (tv)0.762**0.0450.810 0.0780.811 * 0.0810.683*0.081 in a civil union (versus religious)0.7910.1160.8780.2950.7910.1680.9090.248first birth timing versus age 3034age at first birth, 15191.300 0.1701.741 * 0.4081.0030.2031.604#0.429age at first birth, 20241.260 * 0.1321.435 * 0.2691.1970.2001.2340.257age at first birth, 25291.0890.1151.3260.2520.8970.1551.1810.235age at first birth, 35490.383*0.0980.2770.1150.5960.2130.6590.674n women1,8551,855681666508n observations3,1683,1681,2041,175789n events1,3301,330516482332log - likelihood9,123.2449,088.7093,014.9942,819.8851,798.008 * * * p <0.001, * * p <0.01, * p <0.05, #p <0.10 cox regression models for second birth 2001 ilfi survey (all women with a first birth and in a union) * * * p <0.001, * * p <0.01, * p <0.05, #p <0.10 model 2 includes the standard set of covariates as in the models above . Addition of these individual - level traits does virtually nothing to attenuate either the regional or the cohort effects . Women from the northeast and the center display hazard rates that are 21% and 23% higher than women in the northwest, while again, strikingly, women from the south experience the second - child transition at 2.6 times the rate of women in the northwest, even controlling for all of these individual - level traits . Women whose mothers worked when the women were aged 15 show higher rates of second birth transition . While this might be seen as contrary to expectation it may reflect an intergenerational pattern of scheduling work and reproductive careers in which the second birth is hastened by the existence of a maternal role model balancing work and reproduction . Father s education and that of the woman herself carry little predictive value in this model . Those who have their first child at an earlier age also made the transition from first to second child more quickly . Broadly speaking, women who bore their first child before age 25 moved on to the second child at about 25% higher rate than women whose first birth occurred when they were aged 2534 . As in the case of both other transitions considered above, employed women and those in civil marriages exhibit dramatically lower second birth transition rates, with the combined effect of lowering the hazard rate by about 40%.15 as with the models reported for the previous two transitions, models 35 here repeat the analysis by cohort . In the 194150 cohort, regional differences are quite pronounced, with the south relative risk about 2.5 times the northwest and the other two regions intermediate and non - significant . For this cohort of 681 women, about 75% of whom experienced a second birth, those with father s education at the low or high end of the distribution are shown to have slower rates of progression to the second child . Women with limited education show no difference from women with middle - range education, but women with high educational levels have much lower second birth rates (rrr = 0.648). Employment continues to have much the same effect as before, lowering predicted fertility by about 20% . The point estimate for civil union indicates reduced fertility, but civil marriages are rare in this cohort and the estimate does not achieve statistical significance . In the 195160 cohort, women in the south bear their second child at rates about 2.5 times those in the northwest; women in the other regions are intermediate and their differences are not statistically different from the reference region . No parental or personal traits for education or parental loss are statistically significant for this cohort of 666 women, some 72% of whom bear a second child . Working women exhibited rates of childbearing reduced by about one - fifth (rrr = 0.811, p = 0.036). There were no statistically significant differences by union status or by age at first birth, although the presence of this latter group of dummy variables does alter other coefficients . In our youngest cohort, 196170, we have 508 women who initiated a spell with a first birth, and 65% of them are observed to go on to a second birth . Here the regional rankings are similar to earlier cohorts, but we observe particularly depressed fertility among women in the northwest . Southern women have rates of second birth almost four times as large as those in the northwest (rrr = 3.73, p <0.001). Few personal and parental background traits help predict this fertility transition, save one: women s own education . Women in the highest education category exhibit transition rates to second birth 65% above other women . This may represent a conscious fertility compression strategy for such highly educated (and professional) females . Working depresses the rate of transition to the second birth; its effect is significant and larger in magnitude than for previous cohorts . As with the first birth transition, we investigated alternative models for the transition to second birth . Again, we find no statistically significant difference between the effect of part - time and full - time employment . And finally, we again examined a model for an age - education interaction . Here we include dummy variable for the interaction of covariates for woman s own education and her age at first birth across the pooled and cohort - specific estimations.16 once again, the joint test of the group of dummy variable interaction terms was non - significant; thus, the effect of education across women who bear their first child at differing ages is not seen to differ . The italian case has undermined confidence in the two most prominent theories of very low fertility: one that focuses on increases in women s labor force participation, and the other on a shift to less familistic and more secular values . But if we move away from the country comparisons that tend to dominate this literature and instead examine fertility in italy in the context of individual lives, it becomes clear that each of these theoretical approaches has value . At the same time it is particularly interesting to discover that even regional - level comparisons within italy provided little support for either of these two influential theoretical perspectives . It required individual - level life course data and longitudinal analysis to demonstrate that both hypothesized sets of relationships actually do play a role . Indeed, our micro - level analyses showed that women s employment has a strong impact on italians marriage and fertility behavior . Until the most recent cohort, working women married more slowly . And throughout all cohorts, women s participation in the labor force had a consistently negative and powerful impact on their childbearing . Women who were working were 20%30% less likely (using odds ratios from cohort - specific models) to make transitions to first and second birth . Similarly, although the statistical significance was marginal due to the small number of cases, there is intriguing evidence that those choosing a civil marriage and hence embracing more secular values and less subject to the church s influence had lower birth transition rates than those who were married in church . Yet even after controlling not only for women s employment and civil marriage, but also for a host of family and individual characteristics, very pronounced regional effects were evident . This suggests that italy s regional differences are tapping contextual effects that are not captured by existing economic or sdt theory and may be rooted in social networks, provision of local public services, availability of kin, and, especially, sociocultural norms regarding family building . The dramatic size of these regional effects offers strong evidence of the need to look beyond traditional economic and sdt theories in explaining declines in fertility . Contextual covariates, although not easy to assemble, might point to some of the regional and local features both social and policy - based that could help promote or retard these three transitions . Second, one might consider livi - bacci s contention that, contrary to sdt theory, it is the very surfeit of familism in societies such as italy that contributes to very low fertility (livi - bacci and savini 2000; livi - bacci 2001; dalla zuanna 2001). He argues that the intense interdependence between generations is leading to a prolonged period of dependency in the younger generation, and that this leads the younger generation to avoid, postpone, or in any case limit their assumption of parental responsibilities and commitments . Although this theory has proven difficult to test, the argument is provocative and, given the prominent place occupied by familistic societies among the pioneers of very low fertility, worth putting to the test.17 third, it is important to look more closely at gender relations as a potential key to explaining very low fertility . Focusing on gender relations rather than familism, mcdonald has offered an alternative explanation for the italian paradox . 4278) hypothesizes that very low fertility in advanced countries today is the outcome of a conflict or inconsistency between high levels of gender equity in individual - oriented social institutions and sustained gender inequity in family - oriented social institutions . From this perspective, the fact that italy and spain have such low fertility is explained by the preservation of a traditional gender system in the context of expanding labor force opportunities for women . All three of these lines of expansion could flow from (and be consistent with) our work, although developing well - specified empirical tests remains a challenge . Perhaps most interesting for social science theory more generally, our work serves both to recover and to challenge economic and cultural approaches to explaining demographic behavior . While neither prevailing economic theory (flfp) nor sdt theory aimed at explaining very low fertility should be wholly dismissed based on the italian evidence indeed each very likely points at forces that have had an impact on italy s dramatic fertility decline together they only partially explain the italian case . The lessons that italy has to provide for our understanding of very low fertility are still largely untapped, awaiting a fuller exploration of the social, institutional, and cultural matrices in which the decision to have children is made.
Metastasis to the spleen is infrequent but varies greatly from series to series (1.6 to 30%), depending on the thoroughness of examination and selection of cases . The most common primaries are lung cancer, cutaneous malignant melanoma, and breast cancer . Splenic metastasis generally indicates late dissemination of disease, and it is unusual to find isolated splenic lesions from solid organ malignancies, with fewer than 25 cases reported worldwide . We report the case of a woman who presented with a splenic lesion 11 months after radical nephrectomy . A 70-year - old caucasian female presented with a 4-week history of malaise and fatigue . This renal tumour had been incidentally discovered on a follow - up abdominal ct scan she was having following a total abdominal hysterectomy and adjuvant radiotherapy for stage 1c endometrial carcinoma 4 years prior . Histological examination of the renal mass had shown a tumour of conventional clear cell type . There was some invasion of the tumour into the perinephric fat and a segmental branch of left renal vein (stage pt3b). Small deposits of tumours were noted on the surface of the fat resection margin . In light of this and the associated increased risk of recurrence, she received a follow - up un - enhanced ct 6 months post - operatively which showed no recurrence, and was due further urological follow - up prior to her new presentation to the general practitioner 11 months post - nephrectomy with lethargy . At this time she was found to be anaemic (haemoglobin 10.4 g / dl) and had a raised alkaline phosphatase level of 188 iu / l (the remainder of liver function tests were normal). A ct scan revealed an enhancing 6 7 cm necrotic lesion in the lower pole of the spleen, thought to be a metastasis from her previous rcc, with her previous endometrial carcinoma being a less likely primary . It was uncertain whether this was indeed a true metastasis or a local recurrence of her previous left renal tumour . There was no evidence of any other metastatic disease on cross - sectional imaging of the chest and abdomen . After discussion in a multidisciplinary setting, although an uncommon event, the splenic lesion was thought likely to be an isolated splenic metastasis from her previous rcc and resection was recommended . She elected to undergo surgery (open splenectomy), had an uncomplicated procedure / recovery and was discharged from hospital on the eighth post - operative day . Serial slicing revealed a circumscribed centrally necrotic cream - coloured tumour measuring 90 63 55 mm (fig ., two fragments of diaphragmatic tissue were sampled to assess for any evidence of tumour spread . Histological examination of the splenic lesion showed a well - circumscribed, partly encapsulated tumour of solid islands and sheets of cells with an abundant amount of predominantly clear but also eosinophilic cytoplasm (fig . The tumour was found to bulge into the splenic capsule but no definite evidence of capsular breach was found . There was also no evidence of vascular invasion and the lesion was clear of the splenic hilum . No evidence of disease was found in histological samples taken from the diaphragm and therefore the mass was concluded to be a true metastasis of the original rcc rather than local recurrence of the disease . The patient has received on - going follow - up under the care of the urology team, with 6-monthly ct scans which have thus far not shown any evidence of disease recurrence 24 months post - splenectomy . The causes of isolated solid splenic lesions are wide and varied, and as such can present a diagnostic challenge . A splenic mass without any history of malignancy suggests a primary lesion such as lymphoma, vascular tumours or infectious lesions . Any past or present history of malignancy however would lead to a high index of suspicion for a splenic metastasis . In the presence of isolated splenic metastasis interestingly this has been described by mcgregor et al . Who reported a clinically unidentified rcc diagnosed by fine needle aspiration of the spleen . Further differential diagnoses for isolated splenic mass include rarer lesions such as hamartoma and inflammatory pseudotumour, which can both clinically and radiologically mimic a metastatic tumour . One should also consider the possibility of secondary involvement of the spleen through direct invasion, for example from a rcc . Splenic metastases were previously considered exceptionally rare, however more recent advances in radiological imaging and the increased follow - up of patients with cancer means that the rate of detection has increased . An autopsy study found only 8% of 92 patients with secondary non - lymphoid splenic lesions at autopsy were symptomatic . The majority of splenic metastases are part of a multi - visceral metastatic disease, and the detection is often made soon after diagnosis of the primary tumour (mean duration after detection of primary tumour is 6.7 months), however this is often late in the course of disseminated disease . There has however been a case of splenic metastasis 5 years after a radical prostatectomy for prostate carcinoma . This case, along with recent advances in the knowledge of the metastatic process, makes it seem likely that late occurrence of solitary splenic metastases might develop from early blood - borne micrometastasis within the spleen after a period of clinical latency . In an autopsy study of splenic metastasis, the rate of dissemination to the spleen was highest from lung cancer, cutaneous malignant melanoma, and breast cancer, accounting for 24.6, 15.8, and 12.3% of all spleen metastases, respectively . Rcc has been reported to have spread to nearly every organ; however the incidence of splenic metastasis is relatively low, with a 4.6% incidence in an autopsy study . To our knowledge there have been only 8 cases of a splenic metastasis from a rcc that presented clinically and has been reported on, including our study [11, 12, 13, 14, 15, 16, 17]. The incidence of isolated splenic metastasis from any given primary is also particularly rare, with only 5.3% being found to be isolated at autopsy . The reason for the relative rarity of splenic metastases might be explained by two main causes: (1) mechanical factors impeding the splenic implantation of blood - borne cancer cells, such as the constant flow of blood through the spleen and the rhythmic contraction of the splenic capsule, the sharp angle of the splenic artery branching from the coeliac trunk thus preventing large clumps of tumour cells from passing through, and the lack of afferent lymphatic vessels limiting lymphogenic metastases, and (2) the inhibitory effect of the splenic microenvironment on the growth of metastatic cells . Advances in imaging techniques have not only resulted in an increase in the detection of splenic lesions, but also in the cellular diagnosis using percutaneous biopsy (using ultrasound or ct guidance) [7, 19] or endoscopic fine needle aspiration . Given that a splenic metastasis is often found in the context of disseminated malignancy, it tends to have no clinical importance . A splenectomy however can be indicated for palliative symptomatic relief, with reports of acceptable morbidity and improvements in quality of life . They are often asymptomatic and picked up either incidentally or on surveillance imaging for previous malignancy . A splenectomy is an effective and safe option for symptomatic relief and prevention of future complications.
It is a common practice in south east asia to consume alternative medicine which may include toxic metals or animal products for various health reasons . Herein we report a case of anuric acute renal failure with dyspnea and hemoptysis following volitional consumption of rohu fish (labeo rohita) gall bladder . Forty two year old male chronic alcoholic, smoker and marijuana addict presented with history of diffuse abdominal pain with profuse vomiting followed by oliguria . He was treated with intravenous fluids and diuretics in a primary health centre near nagpur . As he developed edema feet, puffiness of face, breathlessness and hemoptysis he was transferred to our tertiary care hospital . There was no history of fever, joint pains, rash, jaundice or bleeding from other sites . On admission, examination revealed medium frame, poorly nourished patient with pallor and no icterus . Investigations revealed hemoglobin-11.4 gm / dl with total white blood cell count-9600/cumm and platelet count-80000/cumm . Urine routine showed protein 2 + with 20 - 25 red blood cells and 70 - 80 white blood cells per high power field . Renal chemistry revealed blood urea nitrogen-160 mg / dl, serum creatinine-16.8 mg / dl, serum sodium-113 meq / l, serum potassium-3.3 meq / l, total proteins-6.6 g / dl with albumin 3.4 g / dl, ast / alt-56/42 iu / l, serum bilirubin-1.1 mg / dl, serum uric acid-11.6 mg, serum calcium-8 mg / dl and serum phosphate-5.5 mg / dl . Ultrasonography of the abdomen showed normal size kidney with no dilatation of pelvi - calyceal system . As patient had hemoptysis and acute kidney injury there was a strong suspicion of pulmonary renal syndrome . However his igg anti - glomerular basement membrane (gbm) antibody was 1.8 u / ml (negative <12 u / ml). A plain computerized tomography (ct) of the chest showed patchy perihilar ground glass opacities with peribronchial cuffing, segmental atelectasis of bilateral lower lobes with bilateral pleural effusion . Persistent enquiry as regards a possible toxic etiology for his acute onset complete anuria revealed he had consumed the entire bile from a raw fish gall bladder (rohu or labeo rohita) a day prior to the onset of symptoms as a cure for his general debility . In view of his pulmonary edema and deteriorating renal function he was dialysed once on admission after which there was a gradual increase in urine output over 6 days . He improved symptomatically with a decrease in serum creatinine from 16.8 mg / dl to 10 mg / dl on day 5 to 4.8 mg / dl on day 15 . He was eventually discharged after 18 days of in - hospital treatment with serum creatinine of 2.5 mg / dl . Subsequent follow - up after 15 days showed serum creatinine was 1.08 mg / dl . Fish gall bladder is considered a traditional medicine to treat rheumatism, decreased visual acuity, and urticaria and to increase sexual vitality . However it can lead to acute renal failure and acute liver injury as reported in cases of fish poisoning from india, japan and hong kong . The ichthyotoxic effects of fish gall bladder is attributed to the presence of a toxin, sodium cyprinol sulfate which is a c27 bile acid . The toxin is heat stable and insoluble in alcohol as cases are reported even after consumption of cooked bile . The family includes grass carp (c idellus), common carp (cyprinuscarpio), silver carp (hypophthalmichthysmolitrix), mylopharyngodonpiceus, labeorohita and aristichthys nobilis . Amongst these, fish of the grass carp variety has been commonly reported for its toxicity but xuan et al . Also reported toxicity secondary to ingestion of shark (minnow) fish (m chrysophekadion) and the bony - lipped barb fish (o melanopi). Even though fish gall bladder can cause severe systemic complications, toxicity does not occur in all cases . In rural vietnam, gall bladder from smaller fish are consumed regularly without evident toxicity . Thus it can be said that toxicity is directly proportional to the size and quantity of gall bladder or bile consumed . Following ingestion almost all patients develop gastrointestinal symptoms within a few hours as reported by xuan et al . In his series of 17 cases from vietnam . Has also reported extra - renal manifestations of the toxin with deleterious effects on the heart and liver with eventual multi - organ dysfunction syndrome . However patients usually seek therapy for renal failure which has an incidence of 55 - 100% . Renal failure is mostly secondary to acute tubular necrosis which may be a direct effect of the toxin . Kidney biopsy has shown the presence of proximal tubular cell damage with focal destruction of epithelial cells on light microscopy . The toxin is believed to damage lysosomes and inhibit cytochrome oxidase thus blocking cellular metabolism, leading to cell necrosis . This in addition to loss of fluid can lead to decreased effective circulating blood volume and eventually renal failure . However the exact mechanism of systemic injury especially renal failure is not known and needs further study . Our patient had reported hemoptysis prior to admission and had an abnormal ct scan of the chest . The cause for the same in such a setting could include fluid overload - pulmonary edema (anuria) or pulmonary renal syndrome . Toxic bile is also reported to cause pulmonary renal syndrome however the patient's primary workup including ana, c3, c4 and igg anti gbm was normal . Toxin induced pulmonary renal syndrome remains a possibility however a workup for the same including kidney biopsy and bronchoalveolar lavave was not done in view of steady improvement in his condition . Most of the reported cases till date have undergone hemodialysis in view of renal failure pandey et al . In their case good awareness programs can aid diagnosis and prevent death from such a dangerous preventable cause of acute kidney injury which is the aim of international society of nephrology 0 by 25 initiative.
, we will provide an update on clinical features, pathogenesis, and treatment options . Clinically nc is characterized by closely arranged, dilated follicular openings with keratinous plugs resembling classical comedones . It is a rare type of an epidermal nevus usually with predilection for the face and neck area . Comedo nevus. Nc can occur congenitally or develop later in life, most commonly at the age of approximately 10 years . Nc is part of the nevus comedonicus syndrome (ncs), a term coined by engber in 1978 [1, 3]. The prevalence of nc has been estimated from 1 in 45,000 to 1 in 100,000, with no gender or racial preference [1, 3]. In 50% of patients, the condition develops shortly after birth and in the majority of patients lesions appear before the age of 10 [46]. Single cases of delayed development of nc and ncs at later age have been reported [46]. Nc is usually asymptomatic and most commonly it affects the face and neck area and, by exception, other anatomical regions, including genital area, palms, and soles . Clinically, nc presents with grouped, dilated, plugged follicular ostia in a honeycomb pattern . Their appearance resembles black dots but the material cannot be easily removed mechanically in contrast to acne comedones [1, 7]. Nc lesions might present with various patterns of distribution: unilateral, bilateral, linear, interrupted, segmental, or blaschkoid (figs . 1, 2 and 3).fig . 1nevus comedonicus: plaque - like lesion with wide open follicles on the neck (44-year - old caucasian male)fig . 2stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings . 3nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian nevus comedonicus: plaque - like lesion with wide open follicles on the neck (44-year - old caucasian male) stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings . A presentation of linear nevus comedonicus with multiple follicular openings . D with compression garment to avoid postsurgical lymphedema nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian two types of nc have been identified; the first is non - pyogenic nc with acne - like characteristics and a second type characterized by formation of cysts, papules, pustules, and abscesses in various stages of development [8, 9]. The former type is also known as munro s acne nevus and separated by some authors from nc . Differential diagnoses are shown in table 1.table 1histopathological differential diagnoses of nevus comedonicusnevus comedonicustrichofolliculomadilated pore of winerpilar sheath acanthomafolliculocystic and collagen comedo hamartomahair cortexlocalization head+++ face++++ neck++ trunk(+)+++ limbs+folliclesnumerous open folliclesopen mother folliclesnumerous open folliclessolitary lesionscysts+++hair shafts+, velluslamellated keratinothersmassive epithelial proliferations without folliclesabundant collagen formation histopathological differential diagnoses of nevus comedonicus the major histopathologic symptoms are large grouped, dilated follicular ostia devoid of hair shafts but filled with keratin layers (fig . One may observe singular rudimentary glands, which are not, however, obligatory as they may be absent . Small cysts, cystic invaginations, and occasionally large cysts may be seen in histopathologic investigation; the variable cystic structures are lined by keratinizing squamous epithelium [1, 7]. Hyperkeratosis (epidermolytic hyperkeratosis) and acanthosis of the epidermis may be present but not para- or dyskeratosis [8, 1013].fig . 4histopathology of nevus comedonicus . A overview (hematoxylin and eosin stain [he] 2). B details with large follicles containing lamellated keratin but absent hair shafts (he 4) histopathology of nevus comedonicus . A overview (hematoxylin and eosin stain [he] 2). B details with large follicles containing lamellated keratin but absent hair shafts (he 4) an increased number of langerhans cells had been observed by electron microscopy . More abundant tonofilaments in the upper part of the stratum spinosum and numerous keratohyalin granules were also noticed [11, 12]. The arrector pili muscles were incompletely differentiated and demonstrated intracellular glycogen particles [11, 12]. Filaggrin, which is usually located in the granular epidermal layer, occurs in the whole epidermis of closed comedones suggesting its role in their development [14, 15]. Interaction between fibroblast growth factor (fgf) and fgf receptor-2 (fgfr2) is a crucial pathway for mesenchymal epithelial interplay in pilosebaceous unit development . Fgfr2b is exclusively found in epithelial cells including epidermal keratinocytes and sebocytes [15, 16]. Overstimulation of fgfr2 signaling with increased expression of interleukin-1 may be involved in nc pathogenesis . Somatic mutations like ser252trp substation have been identified in individual patients with segmental acne [1618]. -secretase found in human hair follicle epithelium is another candidate for nc since the absence of this enzyme results in complete conversion of hair follicles to epidermal cysts . Ncs (orpha64754) belongs to the group of epidermal nevus syndromes including schimmelpenning syndrome, phacomatosis pigmentokeratotica, angora hair nevus syndrome, and becker nevus syndrome among others where a genetic basis has yet not been identified [3, 4, 20]. Ncs is a neurocutaneous disorder which consists of nc with skeletal, ocular, and central nervous system abnormalities . The most common symptoms include cataracts, scoliosis, fused vertebrae, spina bifida, and delayed mental development [5, 6]. In addition, seizures, paresis, dysgenesis of corpus callosum, electroencephalographic abnormalities, limb deformities such as absence of the fifth finger, polysyndactyly or clinodactyly, oligodontia, and other skin disorders such as ichthyosis, leukoderma, lichen striatus, linear morphea, sturge - weber syndrome, hemangiomas, and inverse acne have been reported in ncs [1, 3, 4, 2022]. Ncs is associated with epithelial tumors such as trichoepithelioma, pilar sheath tumor or dilated pore of winer, keratoacanthoma, syringocystadenoma papilliferum, hidradenoma papilliferum, and very rarely basal cell carcinoma or squamous cell carcinoma [2326]. The prevalence of nc has been estimated from 1 in 45,000 to 1 in 100,000, with no gender or racial preference [1, 3]. In 50% of patients, the condition develops shortly after birth and in the majority of patients lesions appear before the age of 10 [46]. Single cases of delayed development of nc and ncs at later age have been reported [46]. Nc is usually asymptomatic and most commonly it affects the face and neck area and, by exception, other anatomical regions, including genital area, palms, and soles . Clinically, nc presents with grouped, dilated, plugged follicular ostia in a honeycomb pattern . Their appearance resembles black dots but the material cannot be easily removed mechanically in contrast to acne comedones [1, 7]. Nc lesions might present with various patterns of distribution: unilateral, bilateral, linear, interrupted, segmental, or blaschkoid (figs . 1, 2 and 3).fig . 1nevus comedonicus: plaque - like lesion with wide open follicles on the neck (44-year - old caucasian male)fig . 2stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings . A presentation of linear nevus comedonicus with multiple follicular openings . 3nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian nevus comedonicus: plaque - like lesion with wide open follicles on the neck (44-year - old caucasian male) stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings . D with compression garment to avoid postsurgical lymphedema nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian two types of nc have been identified; the first is non - pyogenic nc with acne - like characteristics and a second type characterized by formation of cysts, papules, pustules, and abscesses in various stages of development [8, 9]. The former type is also known as munro s acne nevus and separated by some authors from nc . Differential diagnoses are shown in table 1.table 1histopathological differential diagnoses of nevus comedonicusnevus comedonicustrichofolliculomadilated pore of winerpilar sheath acanthomafolliculocystic and collagen comedo hamartomahair cortexlocalization head+++ face++++ neck++ trunk(+)+++ limbs+folliclesnumerous open folliclesopen mother folliclesnumerous open folliclessolitary lesionscysts+++hair shafts+, velluslamellated keratinothersmassive epithelial proliferations without folliclesabundant collagen formation histopathological differential diagnoses of nevus comedonicus the major histopathologic symptoms are large grouped, dilated follicular ostia devoid of hair shafts but filled with keratin layers (fig . 4). At some locations in the bases of the follicular invaginations one may observe singular rudimentary glands, which are not, however, obligatory as they may be absent . Small cysts, cystic invaginations, and occasionally large cysts may be seen in histopathologic investigation; the variable cystic structures are lined by keratinizing squamous epithelium [1, 7]. Hyperkeratosis (epidermolytic hyperkeratosis) and acanthosis of the epidermis may be present but not para- or dyskeratosis [8, 1013].fig . 4histopathology of nevus comedonicus . A overview (hematoxylin and eosin stain [he] 2). B details with large follicles containing lamellated keratin but absent hair shafts (he 4) histopathology of nevus comedonicus . A overview (hematoxylin and eosin stain [he] 2). B details with large follicles containing lamellated keratin but absent hair shafts (he 4) an increased number of langerhans cells had been observed by electron microscopy . More abundant tonofilaments in the upper part of the stratum spinosum and numerous keratohyalin granules were also noticed [11, 12]. The arrector pili muscles were incompletely differentiated and demonstrated intracellular glycogen particles [11, 12]. Filaggrin, which is usually located in the granular epidermal layer, occurs in the whole epidermis of closed comedones suggesting its role in their development [14, 15]. Interaction between fibroblast growth factor (fgf) and fgf receptor-2 (fgfr2) is a crucial pathway for mesenchymal epithelial interplay in pilosebaceous unit development . Fgfr2b is exclusively found in epithelial cells including epidermal keratinocytes and sebocytes [15, 16]. Overstimulation of fgfr2 signaling with increased expression of interleukin-1 may be involved in nc pathogenesis . Somatic mutations like ser252trp substation have been identified in individual patients with segmental acne [1618]. -secretase found in human hair follicle epithelium is another candidate for nc since the absence of this enzyme results in complete conversion of hair follicles to epidermal cysts . Ncs (orpha64754) belongs to the group of epidermal nevus syndromes including schimmelpenning syndrome, phacomatosis pigmentokeratotica, angora hair nevus syndrome, and becker nevus syndrome among others where a genetic basis has yet not been identified [3, 4, 20]. Ncs is a neurocutaneous disorder which consists of nc with skeletal, ocular, and central nervous system abnormalities . The most common symptoms include cataracts, scoliosis, fused vertebrae, spina bifida, and delayed mental development [5, 6]. In addition, seizures, paresis, dysgenesis of corpus callosum, electroencephalographic abnormalities, limb deformities such as absence of the fifth finger, polysyndactyly or clinodactyly, oligodontia, and other skin disorders such as ichthyosis, leukoderma, lichen striatus, linear morphea, sturge - weber syndrome, hemangiomas, and inverse acne have been reported in ncs [1, 3, 4, 2022]. Ncs is associated with epithelial tumors such as trichoepithelioma, pilar sheath tumor or dilated pore of winer, keratoacanthoma, syringocystadenoma papilliferum, hidradenoma papilliferum, and very rarely basal cell carcinoma or squamous cell carcinoma [2326]. Being benign, nc does not require aggressive treatment, except for aesthetic reasons or in complicated cases . Conservative options include emollients and moisturizer, topical corticosteroids (for inflammatory lesions), keratolytics such as salicylic acid, or 12% ammonium lactate solution . Some reports showed better improvement of nc by topical tazarotene as monotherapy or in combination with topical mometasone furoate or calcipotriene [2931]. Oral retinoids, such as isotretinoin, were found to be ineffective in most cases but may be an option in widespread systematized inflammatory variants [32, 33]. There have, as yet, been no published studies of other retinoids, such as topical adapalene and systemic or topical bexarotene . Of potential interest are fgfr inhibitors with antivascular activity . Interleukin-1 inhibitors can be classified into receptor inhibitors, such as anakinra, and monoclonal antibodies against the interleukin itself . Currently, there are no data available for nc . Another interesting drug target is -secretase; stimulators of this enzyme such as general control nonderepressible 2 (gcn2)a subunit of eukaryotic translation factor 2 kinase might be a future therapeutic option . Localized nc can be removed by surgical excision with good aesthetic results in contrast to superficial shaving, comedo extraction, and dermabrasion techniques (fig . If linear nc is surgically removed on the limbs, complex decongestive therapy including manual lymph drainage and compression garments supports the healing process (fig . 2d). Larger lesions may need transplantation for defect closure, or another option is the use of repeat - filling or self - filling osmotic tissue expanders to obtain a resurplus of skin for defect closure [38, 39]. Laser treatments with 2,940-nm erbium yag, 10,600-nm ultrapulsed co2, or 1,450-nm diode lasers have shown improvement in single patients [4042]. Erbium yag laser treatment is often followed by delayed relapses . In contrast to ablative lasers, the 1,450 nm diode shrinks sebaceous glands and reduces seborrhea . The combination of 1,450-nm diode laser with 1,550-nm erbium - doped fiber laser has potential for treating nc . With this review we provided an update on clinical features, pathogenesis and treatment option of nc . Nc is a rare epidermal nevus . New data are available on signaling pathways in acne - related disorders that may have an impact on nc as well . In contrast to the acne - related comedones, the epithelium is often hyperkeratotic and sometimes acanthotic and comedo extraction is not as easily achieved as with acne comedones . Dermatologists should be aware of potential association of nc with skin tumors and extracutaneous findings such as ncs . New developments in laser technology may become an alternative in near future . With a more detailed understanding in molecular pathogenesis, targeted therapy
Several types of studies have suggested that infectious agents may play a role in atherosclerosis and coronary heart disease (chd). Atherosclerosis in chickens is exacerbated by infection with marek's disease virus, a member of the herpes virus family 12, and numerous studies have suggested an association of viral pathogens such as cmv and bacterial pathogens such as helicobacter and chlamydia with human atherosclerosis 345 . Most 678 though not all 910 studies have found that patients with chd are more likely to carry anti - chlamydia antibodies than healthy subjects, and several studies have demonstrated that chlamydia can be detected immunohistologically in> 70% of atherosclerotic lesions, whereas it is virtually undetectable in undiseased arteries 5678 . C. pneuomonia is a gram - negative bacteria that may produce persistent infection through intracellular growth in macrophages 11, a cell type that plays a dominant and necessary role in atherosclerotic lesions 1213 . These observations have led to the hypothesis that chlamydia may play a causal role in atherosclerosis and chd 1415161718 . This hypothesis is made plausible by studies on the association of helicobacter pylori and duodenal ulcer 1920 . This condition was originally thought to be caused by excess gastric acid and was treated by reducing or neutralizing acid . It is now clear that ulcers are most commonly caused by infection with h. pylori, and the most effective treatment involves antibiotics . In the same way, atherosclerosis is currently thought to be caused by excess plasma cholesterol and is treated by agents that reduce cholesterol levels . If chlamydia or any other pathogen causes atherosclerosis, then a more effective treatment may be an antibiotic or vaccine . We have sought evidence for a causal role of infectious agents in atherosclerosis using the murine apolipoprotein (apo) e (deficient) model . Because of the deficiency of apo e, these animals have high levels of plasma cholesterol and develop atherosclerosis with a reproducible time course 212223 . In a first experiment, we bred the apo e with a strain of mouse that is unable to respond to bacterial lps, but found no alteration in the rate or extent of atherogenesis . In a second study we conclude that neither the response to infectious agents nor the presence of infectious agents themselves is absolutely necessary for atherogenesis . Mice with the lps gene crossed onto a c57bl background (c57bl/10scn) were the gift of dr . These mice exhibited minimal increases in serum levels of tnf- and il-6 in response to intraperitoneal injection of 50 g of lps, whereas c57bl and apo e animals exhibited> 100-fold elevations in both (not shown). C57bl/10scn mice were crossed with apo e mice on a c57bl/6 background (jackson laboratories). Apo e/lpsanimals were identified in the f2 progeny by the presence of plasma cholesterol levels> 500 mg / dl and the absence of plasma elevations of tnf- 90 min after injection of 50 g lps . Germ - free and control apo e mice were generated and maintained at taconic farms . Offspring were weaned at 4 wk of age onto a high - fat western - type diet containing 21.22% (g/100 g) fat, 17.01% protein, 48.48% carbohydrate, and 0.15% cholesterol (td88137; harlan teklad) and maintained on the diet for the remainder of the study . Autoclaved food and water were provided ad libitum . The institutional animal care and use committee of merck research laboratories approved the animal use for experimentation, and all animals were cared for in accordance with the guide for the care and use of laboratory animals (1996, national research council). To measure aortic cholesterol and cholesteryl ester, mice were killed and gently perfused through the left ventricle with cold pbs with 5 mm edta . All branches and any adipose tissue connected to the aorta were removed, and each aorta was carefully excised from the aortic root to the right renal artery . The aortas were blotted dry, minced, and extracted with chloroform / methanol (2:1) according to the method used by folch et al . 24 . Total and free cholesterol in the aortic extracts were determined using an enzymatic fluorometric assay modified from previously described methods 2526, and data are expressed on a per aorta basis . Plasma cholesterol and triglyceride levels were determined using standard enzymatic kits (sigma chemical co.). For histology, mouse hearts were perfused in situ with pbs and removed with 1 mm of proximal aorta attached . The top half of the heart was embedded in o.c.t . Embedding medium (fisher scientific) and prepared for cryosectioning . 6-m sections were collected of the aortic root area, defined as having aortic valve leaflets present, and mounted on 10-well masked slides . Additional sections were immunolabeled with mab l3t4 for cd4 or m1/70 for cd11b (pharmingen) using the horseradish peroxidase method with 3 - 3-diamino benzadine as substrate . The time course of atherosclerogenesis in apo e mice was followed by measuring aortic cholesteryl ester (fig . 1), a parameter that tracks with histological progression of atherosclerosis (23; data not shown). To determine a role for gram - negative bacteria such as chlamydia, we first used lps mice 27, animals with a congenital deficiency in responses to the principal inflammatory component of these bacteria, lps (endotoxin). Lps is known to stimulate expression of cytokines (tnf, monocyte chemotactic protein 1 [mcp-1], and others), adhesion molecules (vascular cell adhesion molecule 1 [vcam-1], intercellular adhesion molecule 1 [icam-1], 2 integrins), and enzymes (inducible nitric oxide synthetase [inos], matrix metalloproteinase 9 [mmp9]) observed in atherosclerotic lesions 28 and is a prime candidate to mediate possible proatherogenic effects of chlamydia . Lps mice fail to respond to lps because of a deficiency in toll - like receptor 4 (tlr-4 [29, 30]), a transmembrane protein with homology to the il-1 receptor . The inability of these animals to respond to lps was verified by measurements of plasma tnf in response to lps challenge, and their deficiency in apo e was verified by measurements of plasma cholesterol (described in materials and methods). We observed that development of atherosclerotic lesions in mice fed a high - fat western - type diet was not altered by the deletion of tlr-4 (fig . Further studies showed that plasma cholesterol was also not affected by the absence of tlr-4 (data not shown). This result suggests that responses to gram - negative bacterial products are not critical for murine atherogenesis . To more thoroughly ascertain the role of infectious agents in atherogenesis, we generated a germ - free colony of apo e mice . Founder germ - free apo e pups were delivered by caesarian section and reared by germ - free foster mothers . The colony resulting from these founders was maintained germ free: weekly tests showed that no bacteria could be grown from feces, bedding, or swabs of the isolator . In addition, animals chosen at random showed no antibodies against any of several viral pathogens . The absence of intestinal bacteria is known to cause enlargement of the caecum 31, and dramatically enlarged caeca were observed in all of the animals from the colony . The progress of hyperlipidemia and atherogenesis in germ - free animals was compared with that of a second set of animals taken from the germ - free colony and reared with ambient pathogens . As expected, control apo e animals exhibited extremely high levels of plasma cholesterol and moderately high triglyceride at both 22 and 32 wk of age (fig . Germ - free animals showed an essentially identical plasma lipid profile, and lipid changes are thus unlikely to obscure effects of gnotobiosis on atherogenesis . In both male and female animals, we observed that the exclusion of infectious agents caused no consistent differences in the rate or extent of free cholesterol or cholesteryl ester accumulation in the aorta (fig . 2c and fig . D). We did observe slightly reduced aortic cholesterol ester in the germ - free animals that attained statistical significance at 22 wk of age . However, these small differences disappeared upon correction for reduced body weight observed in the germ - free animals at the 22-wk time point (28 0.6 g for germ - free males vs. 34 0.8 g for control males, and 22 .02 g for germ - free females vs. 24 0.6 g for controls; p = 0.001). The values for aortic cholesteryl ester in germ - free mice were also similar to those seen in a control colony that had never been made germ free (values in fig . The similarity of atherogenesis in control and germ - free animals was confirmed by histologic examination of the aortic root in animals taken at the 22-wk time point . Well - developed lesions with necrotic cores, large foam cells, and fibrous caps were observed in both control and germ - free specimens (fig . B), and neither quantitative nor qualitative differences could be detected in observations of either male or female lesions . A difference in aortic size reflecting the smaller body size of the germ - free animals 31 was readily detected histologically . T cells are found in human and murine atherosclerotic lesions 1223, suggesting activity of the adaptive immune system . We identified t cells by immunohistochemistry in both control and germ - free animals (fig . 3c and fig . D), indicating that pathogens are not required to drive the influx of t cells into atherosclerotic lesions . The observations presented here indicate that infectious agents, whether bacterial, viral, or fungal, are not necessary for murine atherogenesis . Infectious agents do not appear necessary either for initiation or progression of atherosclerosis, nor does the presence of infectious agents alter the morphology or cellular composition of the atherosclerotic lesions . Finally, the presence of infectious agents does not appear to alter the pattern of distribution of lesions along the aorta (not shown). Rather, it appears that the high - circulating cholesterol levels in the apo e animals are sufficient to initiate and drive atherogenesis . We conclude that koch's postulates cannot be fulfilled for any infectious agent in murine atherosclerosis . Atherosclerosis in apo e mice closely resembles that in humans with respect to histology, progression, and dependence on circulating cholesterol 23, and we may thus speculate that infectious agents may not be necessary for the development of human atherosclerosis . We wish to emphasize that the observations reported here relate to atherogenesis, not to plaque rupture, thrombus formation, or myocardial infarction . It is possible that infectious agents play an important role in one or more of these acute processes . Several current studies are seeking to define a role for bacterial infection in human chd by studying endpoints such as myocardial infarction in patients randomized to placebo or antibiotic therapy of varying duration . The largest of these studies to report results thus far has failed to demonstrate a decline in cardiovascular events in the 6 mo after treatment with antibiotic 32 . Although our results suggest that pathogens do not serve as etiologic agents for atherosclerosis, they may still have a role in exacerbating the disease . Recent reports suggest that inoculation of atherosclerosis - prone mice with high doses of chlamydia may cause an approximately twofold increase in the size of atherosclerotic lesions 3334 . It is important to note that stresses such as infection provoke a set of physiological responses that are uniformly proatherogenic 353637 . These include insulin resistance and hyperglycemia, elevation of plasma triglycerides, elevated white blood cell counts, elevated acute phase reactants (e.g., c - reactive protein and fibrinogen), and depression of high density lipoprotein (hdl) cholesterol levels . It is thus possible that infection may exacerbate atherosclerogenesis, and antibiotics may ameliorate atherogenesis not through the action of an etiologic agent, but through indirect effects on metabolism and known risk factors.
Uterine fibroids are the most common uterine neoplasm of uterus and the female pelvis and the most common indication for hysterectomy as well . When the tumor fills the whole pelvis or is pedunculated, there is always an uncertainty regarding the uterine or ovarian origin of the tumor . A cervical fibroid especially with degenerative changes mimics an ovarian tumor and causes a clinical dilemma . A 40-year - old woman, p 2 living 2, presented in the department of gynaecology with the complaint of progressively increasing distension in abdomen for last 1 year . Surprisingly, there were no complaints regarding change of menstrual pattern, urinary or bowel habits, anorexia or fever . On examination, a huge abdominal mass occupying whole of the hypogastrium, both iliac fossa, umbilicus, and left hypochondrium, the mass was 10 8 inches in size, smooth surfaced with regular margin, soft to firm in consistency, nontender, side to side mobile, lower limit not reachable, and engaged in the pelvis more toward the left side . The same mass was felt obliterating all the fornices; uterus was easily palpable of normal size but dextro deviated . Ultrasonography revealed a huge complex mass arising from the pelvis and extending into the abdomen occupying almost whole of the abdomen with well - defined margins; complex internal echotexture, predominantly echogenic with multiple poorly hypoechoic areas along with two cystic areas, each measuring around 4 cm 5 cm in size . A normal size uterus was seen pushed to the right side with normal central endometrium . Clinically provisional diagnosis of the benign ovarian tumor was made based on doppler and biomarker ca 125 levels . After preanesthetic work up, the patient was taken up for laparotomy . Per operatively soft mass of size 12 10 inches with intact external capsule was seen, growing into the leaves of left broad ligament and extending above the umbilicus . Both ovaries, right tube and uterus was normal but left fallopian tube was stretched over the mass . On opening the broad ligament leaf, it was found to be arising from posterior surface of upper part of cervix . Mass was shelled out followed by total hysterectomy with left - sided salpingo - oophorectomy [figure 1]. Cut section showed few solid areas along with multiple cystic areas, one containing black tarry material suggestive of red degeneration . Surgical specimen showing normal uterus with huge cervical fibroid histopathology of the mass revealed benign spindle cell tumor (leiomyoma uteri) with red, cystic, myxoid and hyaline degenerations and dystrophic calcification [figure 2]. Histology: spindle cells tumor (leiomyoma) with cystic, fatty, myxoid, and red degenerations fibroids extending into broad ligament are rare and may mimic an ovarian tumor . It is known that degenerative changes result in unusual appearance that adds to diagnostic confusion . Pedunculated uterine, cervical, and true broad ligament fibroid, especially with degeneration, may be mistaken for lesion of ovarian origin and therefore must be kept in the differential diagnosis . Cervical fibroids pose enormous surgical difficulty by virtue of their relative inaccessibility and proximity to bladder and uterus and distorting the normal anatomical relationships . As in this case it bulges outward between the layers of broad ligament, displacing uterine artery outward and upward and ureter outward toward the pelvic side wall.
Acute lung injury (ali) is a devastating syndrome and an important cause of mortality in critically ill patients . The syndrome is characterized by hypoxemia and respiratory failure due to exudation to alveolar spaces which impairs gas exchange . The risk factors for developing this syndrome include pneumonia, gastric aspiration, sepsis, shock, and acute pancreatitis . Although acute pancreatitis represents one of the less common clinical disorders associated with acute respiratory distress syndrome (ards), severe attacks of pancreatitis are frequently associated with acute lung injury and respiratory failure . The underlying molecular mechanisms behind the development of lung injury are not fully understood, which may explain the lack of specific pharmacologic therapies . Transforming growth factor (tgf-) is a multifunctional cytokine regulating inflammatory and fibrotic disorders . It plays a critical role in embryonic development as well as in the resolution of tissue injury in multiple organs, including the lung . Tgf- exists in three isoforms and is a member of a large family of soluble proteins that modulate several cellular processes . Of these isoforms, tgf-1 is generated in greatest abundance subsequent to tissue damage . Tgf- signaling is initiated via ligand - induced heteromeric complex formation of the tgf- type i and type ii serine / threonine kinases receptors . Upon ligand binding, the tgf- type ii receptor (trii) phosphorylates the type i receptor, which then propagates the signal through phosphorylation of the smad protein cascade . Smad proteins constitute three functional classes: the receptor - regulated smad (r - smad), the comediator smad (co - smad), and the inhibitory smad (i - smad). R - smads (smad1, 2, 3, 5, and 8) are directly phosphorylated and activated by the type i receptor kinases and form heteromeric complexes with the co - smad, smad4 . The activated smad complexes are translocated into the nucleus and, in conjunction with other nuclear cofactors, regulate the transcription of target genes . The i - smad, smad6 and smad7, negatively regulates tgf- signaling by competing with r - smads for receptor or co - smad interaction and by targeting the receptors for degradation . Tgf- has been most thoroughly evaluated for its crucial role in the development of pulmonary fibrosis and airway remodeling during the late phases of chronic lung injury [6, 7]. However, the involvement and regulation of tgf- in acute lung injury are largely unknown . Murine models have demonstrated that the expression levels of several tgf--responsive genes involved in extracellular matrix modulation and fibrinolysis were dramatically increased in the early phase after induction of lung injury . In addition, elevated levels of tgf-1 as well as tgf--inducible genes, such as procollagen type iii and 1, have been demonstrated in the lungs of patients with ards [9, 10]. Procollagen iii is one of the earliest predictors of the severity of ali [1114]. Tgf- has been shown to directly increase alveolar epithelial permeability by increasing the gaps between the endothelial cells [1518]. Increased epithelial permeability permits migration of neutrophils, which stimulates repair of the pulmonary epithelium . We hypothesized that tgf- signaling might be active early in the lungs in ali and plays a significant part in the flooding of the alveolar spaces and lung injury . The aim of the present study was to investigate the early activation of tgf- signaling in the lungs of a murine model of acute pancreatitis - associated ali . Antibodies against tgf-1 - 3, trii, and alk5 (tri) were from santa cruz biotechnology inc . (santa cruz, ca, usa). Antibodies against smad2, 3, 4, and 7 and p - smad2 810 -week- old male wild - type c57bl/6 mice were purchased from charles river, germany . The mice were housed in appropriate facilities at lund university, under specific pathogen - free conditions and handled according to the institute guidelines with approval of the malmo - lund animal care ethics committee . The animals were kept under 12/12 h light / dark regime in standard mesh cages with laboratory chow and drinking water ad libitum . Animal model . Acute pancreatitis was induced using the combined pancreatic duct and bile duct (bpd) ligation model as described previously . The bpd ligation model is a highly acute model that elicits a pronounced pulmonary inflammatory response as early as 9 h after acute pancreatitis induction . Briefly, the mice were anesthetized and maintained with 24% isoflurane . Under aseptic conditions, the bile duct, proximal to its entry into the pancreas, and the common bile - pancreatic duct, near its junction with the duodenum, were dissected and ligated (bpd group). The same procedure was applied to sham - operated control mice where the common bile - pancreatic duct and the bile duct were dissected, but not ligated, after which the abdomen was closed . The mice recovered rapidly after surgery, and postoperative buprenorphine analgesia (0.05 mg / kg, s.c .) Was administered twice daily . The animals (n = 8 in each group) were sacrificed by exsanguination through puncture of the abdominal aorta 9 and 24 h after pancreatitis - induced surgery . Lung biopsies were harvested, fixed in 4% paraformaldehyde for further immunohistochemical processing or snap - frozen in liquid nitrogen, and stored at 80c until western blot analyses . Endogenous peroxidase activity was quenched for 30 min in darkness by 1% h2o2 (sigma aldrich, st . Louis, mo, usa) in methanol (histolab, gothenburg, sweden). After blocking with avidin - biotin (vector laboratories, burlingame, ca) and normal goat serum (5%) for 60 min, the sections were incubated with primary antibodies in tris - buffered saline (tbs)/saponin buffer overnight . Biotinylated goat anti - rabbit igg secondary antibodies were added in 2% normal blocking serum . Slides were developed for 5 min in diaminobenzidine (dab, vectastain, vector laboratories) and counterstained with mayer's hematoxylin (histolab). Positively stained cells were quantified as average number of cells in 10 microscopic fields (20x magnification) by two researchers blinded to the samples . Snap - frozen lung tissue was homogenized in liquid nitrogen using a mortar and pestle . Proteins were extracted in 1% tritonx-100, 10 mm tris - hcl, 50 mm nacl, 5 mm edta, 30 mm sodium pyrophosphate, 50 mm naf, 0.1 mm na3vo4, and complete protease inhibitor cocktail (sigma). Lysates were dissolved in laemmli buffer, boiled and separated by sds - page (12%), and transferred to 0.2 m hybond - c extra nitrocellulose membrane (amersham biosciences). Blots were blocked in 5% (w / v) milk in tris - buffered saline tween-20 (tbst) and incubated overnight at 4c with the primary antibodies (1 g / ml). Following washing, the membranes were incubated for 1 h with the secondary hrp - linked goat anti - rabbit antibody at 1: 2000 . Immunoblotted proteins were visualized by supersignal west dura substrate (pierce thermoscientific) using li - cor odyssey fc imager (li - cor biosciences) and image studio vr 2.0 software . Statistical analyses were performed by two - tailed student's t - test using prism software . A p value of <0.05 was considered statistically significant . To investigate the role of the tgf- system in the progression of ali due to acute pancreatitis, levels of tgf-1, -2, and -3 were examined in the lungs of ligated animals with acute pancreatitis compared to sham . In the lungs of the animals with ap, tgf-1 was associated predominantly with the bronchial epithelium and macrophages at both time points . Occasional fibroblast - like cells and type ii alveolar epithelial cells were also stained (figure 1(b)). There was a 1.68-fold enhanced expression of tgf-1 in the lungs of this group compared to the sham - operated animals (p <0.05; figures 1(a), 1(b), 1(g), and 1(h)). These changes were more pronounced after 24 h as compared to 9 h (p <0.01; figures 1(g) and 1(h)). Staining for tgf-2 in airways of the animals with ap showed similar pattern to that for tgf-1 . Bronchial epithelial cells and smooth muscle cells were stained more intensely compared to other cell populations and fibroblast - like cells were stained more consistently compared to tgf-1 . Neutrophils were positive for tgf-2 as well as macrophages, sub - epithelial fibroblast - like cells which were stained moderately . No obvious difference between the sham - operated and ligated animals at any of the time points (figures 1(c) and 1(d)). Macrophages, fibroblast - like, and smooth muscle cells were also showing expression . In the ligated group, macrophages were generally stained; subepithelial fibroblast - like cells showed mixed but predominantly positive staining . The bronchial epithelial cells showed a stronger staining tendency in the ligated group in both 9 (figures 1(e) and 1(f)) and 24 h (data not shown). Protein extracts from the lungs of the sham - operated and ligated animals were analyzed by western blotting . Total tgf-1 protein levels were higher in the lung extracts of the ligated animals at 9 and 24 h compared to the lungs of the sham - operated group (figures 1(g) and 1(h)). There was not any difference for tgf-2 and 3 protein levels between the sham - operated animals and the acute pancreatitis group (data not shown). These results indicate that early modulation of tgf- ligands in the lungs of mice with acute pancreatitis mostly relates to induction of the tgf-1 isoform rather than tgf-2 and tgf-3 . To assess modulation of two candidate receptors for tgf- signaling, the lung sections were stained for trii and alk5 (tri). Both the acute pancreatitis (figure 2(b)) and sham - operated groups (figure 2(a)) demonstrated high levels of trii in bronchial epithelial and smooth muscle cells, while alveoli, fibroblasts, endothelial, and infiltrating cells showed moderate to weak staining (figures 2(a) and 2(b)). No obvious difference in the expression pattern of trii was observed between the acute pancreatitis group and sham controls at the time points studied . Bronchial epithelial and vascular endothelial cells were strongly positive for alk5, while fibroblasts and alveoli were moderately positive for alk5 in the lungs of the pancreatitis - induced group (figures 2(d) and 2(f)). A similar staining pattern was observed in the lungs of the sham - operated groups (figures 2(c) and 2(e)). Interestingly, the bronchial epithelial cells demonstrated an enhanced nuclear accumulation of alk5 after 9 h that was further enhanced at 24 h after acute pancreatitis induction compared to sham controls . In addition, acute pancreatitis following bpd - ligation was further associated with enhanced number of infiltrating cells that were more alk5-positive compared to sham (figures 2(d) and 2(f)). A significant overall increase in the number of alk5-positive cells per field was found in the pancreatitis - induced groups compared to sham at both 9 and 24 h (figure 2(g), 53 versus 32; p <0.001 and 45 versus 32; p <0.05; resp . ). The elevated alk5 levels in the lungs following acute pancreatitis induction were further confirmed by western blot of total protein extracts . A pronounced increase in the total protein levels of alk5 was detected at both 9 and 24 h in the pancreatitis group compared to sham control (figure 2(h)). These data indicate that the acute pancreatitis mediated regulation of tgf- responses at the receptor level predominantly involves induction of alk5 instead of trii . Smad2 was detected at moderate levels in bronchial epithelium, infiltrating cells, and fibroblasts, while low levels were observed in alveoli and vascular endothelial cells in both sham controls and 9 h after acute pancreatitis induction (figures 3(a) and 3(b)). High levels of smad3 were observed in bronchial epithelium, vascular endothelium, infiltrating, and fibroblast - like cells, while low levels were detected in alveolar cells in both sham controls and with acute pancreatitis induction (figures 3(c) and 3(d)). Similarly, lungs from both sham controls and acute pancreatitis - associated ali showed moderate levels of smad4 in bronchial epithelial, vascular endothelial and infiltrating cells (figures 3(e) and 3(f)). A moderate smad4 induction was noted in the infiltrating cells in the ligated animals compared to sham controls (figure 3(f)). However, neither immunostainings nor western blot (figures 3(g) and 3(h)) analyses showed a significant modulation of the smad24 protein levels in the lungs following 9 or 24 h acute pancreatitis induction compared to sham controls . To further study regulation of the tgf- signaling pathway, bronchial epithelial cells as well as vascular endothelial, smooth muscle, and inflammatory cells showed considerably higher levels of smad7 9 h after acute pancreatitis induction compared to sham controls (figures 4(a) and 4(b)). However, the smad7 induction appeared transient and with disease progression the levels of smad7 were reduced . After 24 h the levels were not altered notably compared to control (figures 4(c) and 4(d)). Analysis of the smad7 protein levels in lung extracts confirmed the immunohistochemistry findings, with 2.41-fold increased levels of smad7 (p <0.05) in the ligated group at 9 h, but not at 24 h, compared to sham controls (figure 4(e)). In order to study the level of active tgf- signaling during acute pancreatitis - associated acute lung injury, the activation and subcellular distribution of phosphorylated smad2 (p - smad2) bronchial epithelial, vascular endothelial, infiltrating, and fibroblast - like cells in the sham - operated animals had a weak cytoplasmic expression of p - smad2 at both 9 and 24 h, with scattered cells showing a nuclear localization (figures 5(a) and 5(c)). In contrast, enhanced levels of cytoplasmic and importantly nuclear translocated p - smad2 were detected in the lungs, especially in vascular endothelial cells, 24 h after acute pancreatitis induction compared to sham control (figures 5(c)5(f)). Despite differences at the cellular level, the total protein level of p - smad2 was not altered between the sham - operated and ligated animals at any of the time points (figure 5(g)). A summary of the changes in expression of the tgf- signaling molecules is presented in table 1 . Tgf- has a well - established role in the fibrotic processes during chronic lung diseases . In the present study, we show that tgf- signaling modulation starts as early as 9 h in the lungs of animals with acute pancreatitis - associated acute lung injury . These changes included an early increase in the levels of tgf-1 and alk5 (tri). A parallel increase in the signaling regulator and general inhibitor of the tgf- signaling transduction, smad7, was found at 9 h. later in the course of the disease at 24 h the levels of tgf1 and alk5 remained high, while levels of the inhibitory smad7 were reduced back to the level of the sham - operated animals . These changes were associated with an enhanced nuclear translocation of phosphorylated smad2 indicating an active tgf- signaling . This is the first report on the early modulation of tgf- signaling pathway in the acute lung injury due to acute pancreatitis . Tgf- signaling regulates various cellular processes, including cell proliferation, recognition, differentiation, apoptosis, and specification of developmental fate during embryogenesis as well as in mature tissues . Previous studies have indicated that tgf- not only participates in the late phase of acute lung injury, but also might be active early in acute lung injury and potentially could contribute to the development of pulmonary edema . Tgf- mrna level increased in murine intraparenchymal mononuclear cells and in alveolar macrophages within 1 h in a hemorrhage - induced acute lung injury model . In addition to its classical role in fibrotic and extracellular matrix remodeling, tgf- signaling has a pivotal role in angiogenesis and vascular defects . The in vitro effect of tgf- on the integrity of vascular endothelium started within 1 - 2 h and was maximal 8 - 9 h after exposure to tgf- . Tgf-1-induced lung endothelial cell barrier dysfunction is mediated by activation of rho and rho - kinase, which induce endothelial cell permeability . This effect appears to result from endothelial cell contraction through the activation of a myosin light chain kinase - dependent signaling cascade . Our results showed an early increase (9 h) in the protein level of tgf-1 in the lungs of animals with acute pancreatitis . This can be a contributing factor to the lung injury due to tgf- effects on the vascular integrity . We have previously shown a significant enrichment of inflammatory cells (neutrophils and macrophages), recruited into the lung tissue of animals with acute pancreatitis at 9 h . In addition to its effects on vascular permeability, tgf-1 has been shown to enhance monocyte migration . Tgf-1-driven monocyte motility was mediated via alk5-induced pi3 k and p38 signaling, independently of smad2/3 . The early rise in both tgf-1 and alk5 observed herein may thus contribute to stimulate the monocyte enrichment in the lungs at 9 h. disruption of the vascular integrity is not the only way that tgf- signaling can play a role in the initiation and progression of acute lung injury . Tgf- activation plays a role in the development of acute lung injury through alteration of fibroproliferative responses, lung permeability, and inflammatory cell influx [18, 2729]. Tgf- and tgf--inducible genes are able to modify lung permeability, epithelial ion transport, fibrinolysis, extracellular matrix, and surfactant homeostasis . The integration of the changes in these molecular pathways by tgf- implicates it as a central mediator of acute lung injury . Mice treated with anti - tgf-antibodies showed decreased levels of pro - inflammatory cytokines compared to untreated one in a hemorrhage - induced acute lung injury model . Different human diseases have been associated with dysregulation of tgf- signaling, which shows the importance of a well - regulated signaling process . Smad7 is part of an autoinhibitory feedback control, which interacts with alk5 through various mechanisms . Smad7 resides in the nucleus in unstimulated cells and translocates to the plasma membrane following receptor activation, where it binds the receptors and inhibits further signaling . Smad7 has a central role in pathological processes by its antifibrotic and anti - inflammatory properties . The anti - inflammatory activities of increased smad7 at early phase in our model can be interpreted as a protective mechanism in the lungs of the animals with acute pancreatitis . In this study, we showed increased levels of both tgf-1 and alk5 in the lungs as early as 9 h after the induction of acute pancreatitis . However, increased tgf- signaling via the smad2 pathway was observed first after 24 h following acute pancreatitis induction . Increased nuclear translocation of phosphorylated smad2 at 24 h can be due to the decreased smad7 levels in the lungs . This time point was the peak of the inflammatory response associated with inflammatory cells recruitment to the lungs as well as pathological changes indicating the acute lung injury, in a previous report . The later activation of the signaling pathway can be of interest in the acute lung injury, making the tgf- pathway a potential therapeutic target . This is in accordance with previous report of attenuated lung injury by administration of a tgf- inhibitor 24 h after the injury . In summary, the present study shows an early rise in tgf-1 and alk5 which may activate the tgf-/smad2 signaling or alternative pathways to contribute to ali . Acute lung injury is a critical syndrome due to a variety of precipitants, including acute pancreatitis . Improvements in outcome following ali over the past decade are in part due to improved strategies of mechanical ventilation and advanced support of other failing organs and not a specific treatment . Clinical and experimental studies indicate a role of tgf- in the early phases of ali, where tgf- contributes to the lung injury through epithelial and endothelial injuries . Our data showed an early rise in smad7 which can block the tgf-/smad2 signaling . With disease progression, the smad7 levels were reduced with a concomitant increase in nuclear translocation of phosphorylated smad2, an indicator of active tgf-/smad2 signaling.
Gonorrhea continues to be a public health problem with the emergence of multidrug resistant strains . Accurate diagnosis with effective treatment to prevent further transmission is one of the essential elements for control of gonococcal infections . Treatment strategies should be devised to utilize appropriate and preferably single - dose therapy that could be conveniently administered at the time of diagnosis . Surveillance of neisseria gonorrhoeae antimicrobial resistance is crucial in guiding empirical therapy in any individual setting as resistance may vary in different countries . Neisseria gonorrhoeae infections have been reported from pakistan as a cause of sexually transmitted infections, but unfortunately national surveillance data is not available to assess its prevalence or antimicrobial resistance [5, 6]. Due to limited resources and lack of trained technologist and microbiologists, therefore, existing antimicrobial resistance data from the country is limited and mainly laboratory based . Previously high resistance rates to penicillin, tetracycline, and quinolone have already been reported from the country [7, 8]. Thus ongoing surveillance of resistance is important considering the public health importance of gonococcal infections . In this study, we have evaluated antimicrobial resistance in neisseria gonorrhoeae for the years (20072009) and compared it with previously reported data (19922006). Additionally, this study was conducted in the clinical laboratory of the aga khan university hospital (akuh), karachi, pakistan . The akuh laboratory is accredited with the joint commission of international accreditation (jcia) and routinely participates in the external quality assurance programme of college of american pathologists (cap). The laboratory has a well - established national specimen collection network with more than 175 collection units located in major cities and towns across the country . Specimens are requested by physicians as per their judgment and are processed in the clinical laboratory based in karachi . The laboratory data presented in this study although was not collected through a programmed survey, but represents strains prevalent across the country . Information regarding the age, sex, year of isolation, and specimen type was retrieved from the computerized database . All isolates in 20072009 were saved in glycerol phosphate broth and stored at 80c . During the study period, antimicrobial susceptibility testing was performed and interpreted according to clinical laboratory standards institute (clsi) criteria using the disk diffusion methodology . All isolates were tested against penicillin (10 g), ceftriaxone (30 g), tetracycline (30 g), and ofloxacin (5 g) (oxoid) on gc agar base and 1% defined growth supplement . Minimum inhibitory concentration (mic) of ofloxacin was determined in strains isolated during 20072009 by the e - test method (ab biodisk, sweden) on gc agar base and 1% defined growth supplement . Additional antibiotics tested in selected strains isolated during 20072009 included cefotaxime (30 g), cefoxitin (30 g), cefuroxime (30 g), cefipime (30 g), ceftazidime (30 g), ceftizoxime (30 g), cefixime (30 g), cefpodoxime (30 g) spectinomycin (100 g), and azithromycin (15 g) (oxoid). The antimicrobial susceptibility of all these antibiotics was interpreted according to clsi criteria except azithromycin that was interpreted using british society of antimicrobial chemotherapy (bsac) methods for antimicrobial susceptibility testing . Strains resistant to penicillin, tetracycline, and ofloxacin were categorized as having combined resistance to penicillin, tetracycline, and ofloxacin . Data extracted from the computerized information system were transferred to the statistical software spss version 14.0 . Chi - square for trend analysis was also conducted to assess resistance trend over the study period . . Majority (93%) of organisms were isolated from urethral, high vaginal, and cervical swabs, (6%) were from pus aspirates, and (1%) were from eye swabs, blood, and urine . During the study period, an increasing trend of resistance was observed against penicillin, tetracycline, and ofloxacin (p - value for trends <0.01) (table 1). N. gonorrhoeae strains with combined resistance to penicillin, tetracycline, and ofloxacin increased from (0%) in 1992 to (70.8%) in 2009 (p - value for trends <0.01). No resistant strains to ceftriaxone were picked up by the disk diffusion method during the 15-year period . Comparison of resistance rates during years 19922006 and 20072009 revealed significantly increased resistance between the two periods (figure 1). Susceptibility to cefotaxime, cefoxitin, cefuroxime, cefipime, ceftazidime, ceftizoxime, cefixime, cefpodoxime, spectinomycin, and azithromycin was determined in 100 isolates . Resistance against the above - mentioned agents was not identified, and 100% of the tested isolates were found susceptible . The results of our study demonstrated increasing trend of resistance in n. gonorrhoeae against penicillin, tetracycline, and ofloxacin in pakistan . The resistance rates reported in this study against first - line drugs markedly exceed the world health organization cutoff of 5%, precluding their use for the empirical therapy in gonococcal infections . Accurate information on antimicrobial susceptibility of prevalent n. gonorrhoeae is, thus, essential for the treatment and control of this disease . Keeping in view the current scenario in pakistan and absence of a national guideline for treatment of gonococcal infections, the selection of appropriate antibiotics for empirical treatment of gonorrhea is challenging . This 10-year analysis clearly showed that the level of resistance to traditional antibiotics used in the treatment of gonorrhea continued to increase in local isolates . This trend follows the global pattern of antibiotic resistance in n. gonorrhoeae and leaves third - generation cephalosporins as the recommended antibiotic for its treatment . However emergence and spread of strains with reduced susceptibility to ceftriaxone is another emerging issue . Unfortunately, these strains are simultaneously resistance to multiple drug classes, including quinolones, macrolides, penicillins, and tetracyclines . We did not perform ceftriaxone mic, required to detect reduced susceptibility, and there is a possibility to miss their occurrence as the frequency of mdr strains constitutes a large proportion of our current n. gonorrhoeae isolates circulating in the community . On the other hand, treatment failure with ceftriaxone the best strategy in this scenario would be periodic determination of ceftriaxone mics to detect existence of these strains and close followup with clinicians to identify treatment failure . A high quinolone resistance approaching almost 100% in recent years was demonstrated in local isolates . This exponential rise in quinolone resistance has also been reported from the region and raises several concerns [15, 16]. We had not evaluated the exact reason for this finding, but probable reasons could be its continuous use despite higher rate of resistance and poor disease control in the community . Increased resistance rates to quinolones have been reported from the country in other community acquired pathogens highlighting the overall misuse of quinolones as the major reason of increased resistance [17, 18]. In view of high resistance to the currently tested antimicrobials in our setting, we tested 8 cephalosporins including 3 oral options: cefixime, cefpodoxime, and cefuroxime . Although cefpodoxime and cefuroxime have shown a cure rate of more than 95%, their pharmacodynamic parameters are less favorable than those of cefixime and ceftriaxone [20, 21]. Nevertheless, they could be used as oral alternatives in the treatment of uncomplicated gonorrhea in our setting . Cefotaxime, cefoxitin, cefepime, ceftazidime, and ceftizoxime are the other single - dose cephalosporins included in the updated treatment regimens for gonococcal infections by the centers for disease control and prevention . Although studies have reported emergence of resistance to these agents, we were not able to detect resistance against any of them in this study . Local isolates were also uniformly susceptible to spectinomycin, which is a good option especially in those patients who cannot tolerate cephalosporins . The most likely reason for its consistent susceptibility is its less - frequent use in the community . Rapid emergence of resistance has been reported with the wider use of spectinomycin mandating its rationale use . Though azithromycin is so far not recommended as a single agent for the treatment of gonorrhea, it has a potential role in the management of these infections in combination with third - generation cephalosporin . We did not detect resistance to this drug although strains with high level resistance have emerged as another threat [25, 26]. Although our dataset included samples from all over pakistan, sampling limitations prevent us from generalizing our results to the entire population of the country . However, it is important to note that only a few laboratories in the country perform antimicrobial susceptibility testing in neisseria gonorrhoeae, and our data is the largest ever from the country reporting resistance over time . Another limitation is lack of mics determination for cephalosporins with possibility of missing strains with reduced susceptibility . In conclusion, we recommend that penicillin, ciprofloxacin, and tetracycline should not be used in the empirical treatment of gonorrhoeae . Ceftriaxone and cefixime should be the first line therapy; however, periodic mics should be determined to identify emergence of strains with reduced susceptibility . Optimized, standardized, and quality assured antibiotic susceptibility testing needs to be established in laboratories in pakistan . Simultaneously prevention strategies should be strengthened to maximize the clinical utility of these antimicrobials . Such strategies could include the use of a limited number of treatment regimens and overall avoiding a general misuse of the remaining yet effective antibiotics . In addition, a national policy for management of gonococcal infections should be developed and disseminated to clinicians in the country.
Work done in numerous laboratories has demonstrated that stem cell administration is able to reduce the loss of function in the heart after myocardial infarction . We have demonstrated that intravenous injection of bone marrow mesenchymal stem cells (msc) into mice one hour after coronary artery occlusion is able to attenuate the loss of function due to myocardial infarction . These msc secrete the paracrine factors vascular endothelial growth factor (vegf), monocyte chemotactic protein-1 (mcp-1), macrophage inflammatory protein-1 (mip-1), and mip-1 which may play a role in the protective effect of msc [2, 3]. Understanding the way in which the synthesis and secretion of these factors are controlled is important to better utilize msc in clinical situations . The amount of oxygen present while msc are cultured can have a significant effect on the behavior of msc . The atmospheric oxygen level routinely used for culture (21% o2; normoxia) is significantly elevated compared to the 12% oxygen found in arterial blood and the 17% found in a variety of tissues like skin, bone marrow, myocardium, brain, and spleen [47]. In addition, after permanent coronary artery occlusion in rats, myocardial oxygen partial pressure levels drop from 15 mmhg (2%) to less than 2 mmhg (<0.3%) which is maintained after left ventricular remodeling . Hypoxia, or in situ normoxia, alters msc gene expression, affects the secretion of paracrine factors [1113], and influences the ability of msc to differentiate [1416]. Similarly, the amount of serum present in the culture media affects stem cell behavior . Msc are generally cultured in 1020% serum which contains numerous factors that may not be present in the tissues where these cells reside . In addition, the ischemic conditions present after coronary artery occlusion include both hypoxia and serum deprivation [17, 18]. Reduction of serum levels to 2% promotes cardiomyocyte differentiation, reduces cell proliferation, and upregulates genes for maintaining stemness, angiogenic factors, and endothelial differentiation in msc . Serum deprivation alters the secretion of paracrine factors and the expression of stem cell and endothelial markers in msc [2123]. We have previously demonstrated that msc secrete vegf, mcp-1, mip-1, and mip-1 which have significant biological effects on cell migration, apoptosis, and capillary formation . Therefore, the purpose of the present study was to mimic the conditions present during myocardial ischemia by lowering oxygen and serum concentrations during culture in order to determine the effects of these conditions on the gene expression and secretion of vegf, mcp-1, mip-1, mip-1, and mmp-2 by bone marrow msc . To that end, we cultured msc in 5% or 20% serum in either a normoxic (21% o2) or hypoxic (1% o2) environment . Murine mesenchymal stem cells (msc) were isolated from bone marrow and cultured in mesencult basal medium with 20% murine serum supplement (stem cell technologies) as described previously . Msc (passages 310) were cultured at 37c in 6-well plates in 2 ml / well mesencult with 5% or 20% murine serum supplement at a concentration of 0.30 10 cells / well . Normoxic cells were cultured for 48 hours under normal atmospheric oxygen (21%) plus 5% co2 . Hypoxic cells were cultured for 24 hours under normal atmospheric oxygen and then for an additional 24 hours in a reduced oxygen atmosphere (1% o2, 5% co2, 94% n2). A cell proliferation assay using alamarblue (invitrogen; dal1025) was performed in order to correct for differences in growth rates under the different culture conditions . After 48 hours of culture, the wells were washed with pbs and then treated with mesencult + 5% serum supplement containing 10% alamarblue reagent . Absorbance was measured at 570 and 600 nm and the amount of alamarblue reduction (ar) was determined . After 48 hours of culture, media were removed and centrifuged at 14,000 g for 10 min . The supernatant was removed, separated into single use aliquots, and stored at 20c . Media supernatants were analyzed by elisa using quantikine kits (r&d systems) for vegf (mmv00), mcp-1 (mje00), mip-1 (mma00), mip-1 (mmb00), and matrix metalloproteinase-2 (mmp-2; dmp2f0) according to manufacturer's directions . In order to correct for differences in cell proliferation and thus the amount of secretion produced per cell, all values were normalized to those measured for cells cultured in 5% serum under hypoxic conditions using the normalization factors determined by the alamarblue cell proliferation assay (see below). Total rna was isolated after 48 hours of culture using rnaqueous-4pcr kits (ambion). Rna was treated with dnase i and then quantified and assessed for quality by measuring absorbance at 260 and 280 nm . Reverse transcription was performed on 1 g rna using an iscript cdna synthesis kit (biorad) with blended oligo (dt) and random primers . Qpcr was done on 2 l rt dna template with iq sybr green supermix (biorad) using the biorad mj mini thermal cycler with miniopticon with 300 m specific primers (table 1). After an initial hot start at 95c (3 minutes), 40 cycles of 95c (30 seconds), 55c (30 seconds), and 72c (1 minute) were performed . Single band products of the appropriate molecular size were confirmed for the primers using 2% agarose electrophoresis . Relative expression was determined using cfx manager (biorad) by comparing data to the reference gene ywhaz (tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, zeta polypeptide); this was then normalized to the average expression level . Mean and standard error were calculated for each treatment group, and significance was determined using student's t - test the following biologically relevant comparisons were made: (1) normoxic cells grown in 5% and 20% serum, (2) hypoxic cells grown in 5% and 20% serum, (3) normoxic cells with hypoxic cells grown in 5% serum, and (4) hypoxic cells with normoxic cells grown in 20% serum . Before the amount of paracrine factor secretion could be compared between treatment groups, differences in the number of cells present after 48 hours of culture had to be equalized . It was found that cells cultured with 20% serum supplement had significantly elevated levels of alamarblue reduction (ar) (p <0.01; n = 5) compared to 5% serum . While hypoxia had no effect on cells cultured in 5% serum, mean ar values were elevated in hypoxic conditions in 20% serum (p <0.05; n = 5). Compared to cultures in 5% serum under normoxic conditions, the ar value was 24% higher in 20% serum / normoxic cultures and 36% higher in 20% serum / hypoxic cultures . Based on this data, elisa values for 20% serum / normoxic cultures and 20% serum / hypoxic cultures were normalized to 5% serum / normoxic cultures by multiplying by 0.804 and 0.737, respectively . No correction factor was used for 5% serum / hypoxic cultures since ar values were not significantly different from 5% serum / normoxic cultures . The secretion of paracrine factors by msc into the culture media was quantified using elisa (table 2, figure 1). Serum reduction from 20% to 5% and changing from a normoxic to a hypoxic environment had significant effects on levels detected in the media . Previous studies in our laboratory have demonstrated that vegf, mip-1, mip-1, and mcp-1 were not detectable in mesencult media containing 20% serum supplement . Vegf levels were significantly reduced (p <0.01) in media from cells cultured in 5% serum supplement compared to those in 20% supplement in both normoxic and hypoxic conditions . As seen with vegf, serum reduction to 5% significantly reduced (p <0.01) mcp-1 levels in media compared to those in 20% supplement in both normoxic and hypoxic conditions . In addition, hypoxia significantly reduced (p <0.01) mcp-1 levels in both 5% and 20% serum cultures . Mip-1 levels were unaffected by serum supplement concentration or oxygen levels . On the other hand, mip-1 levels (table 2, figure 1) were significantly elevated (p <0.05) in 5% serum compared to 20% in hypoxic conditions, an effect not seen in normoxic conditions . Also, hypoxia significantly reduced (p <0.05) mip-1 secretion in both 5% and 20% serum supplements . Changes in mmp-2 secretion followed the general trends seen for mip-1. Serum reduction to 5% resulted in a significant elevation (p <0.01) of mmp-2 levels compared to 20% in both normoxic and hypoxic conditions . Also, hypoxia significantly reduced (p <0.01) mmp-2 secretion in both 5% and 20% serum cultures . The normalized relative expression of mrna in msc for the various paracrine factors was determined using rt - qpcr (table 3, figure 1). Serum reduction to 5% caused a significant drop in the relative expression of vegf mrna compared to 20% in normoxic (p <0.05) but not in hypoxic conditions . Hypoxia resulted in a significant elevation of vegf expression levels for cells grown in 5% serum (p <0.05%) but not in 20% . This is similar to the response observed with vegf secretion into the surrounding media except for the expression levels in 5% serum . Serum reduction to 5% had no significant effect on the relative expression of mcp-1 mrna compared to 20% serum supplement . Hypoxia significantly reduced (p <0.01) the relative expression of mcp-1 mrna in both 5% and 20% serum supplement . This was similar to what was observed for mcp-1 secretion except for the lack of an effect of serum . Changes in the relative expression of mip-1 and mip-1 mrna were similar to each other . Expression was significantly reduced (p <0.05) in 5% serum compared to 20% in hypoxic conditions . Hypoxia significantly elevated mrna expression in both 5% (p <0.05) and 20% (<0.01%) supplements . These effects were in contrast to secretion of these two factors into the culture media where hypoxia had no effect on mip-1 and suppressed mip-1 secretion . Reducing the serum from 20% to 5% significantly increased (p <0.05) the relative expression of mmp-2 mrna in normoxic conditions similar to secretion into the media . However, reducing serum had no significant effect in hypoxia in contrast to secretion which showed an increase . Hypoxia had no effect on expression in either serum concentration while it reduced secretion into the media . This study clearly demonstrates that alteration of oxygen concentrations and serum levels results in significant changes in the mrna expression for and secretion of paracrine factors by msc . It is the first time that these two parameters have been studied simultaneously and the data suggests that the low serum and oxygen conditions present during the ischemia found after coronary artery occlusion may have significant effects on the secretory function of msc and the role they play in preventing the loss of myocardial function after heart attack . We found that hypoxia significantly reduced the gene expression for mcp-1 but significantly increased the expression of genes for mip-1, mip-1, and vegf (5% serum only) while mmp-2 expression was unaffected . Secretions of mcp-1, mip-1, and mmp-2 were all significantly reduced by hypoxia while vegf and mip-1 were unaffected . The reasons for the differences between mrna expression and secretion for mip-1, mip-1, and mmp-2 are not immediately apparent but may be due to differences in the control of gene expression versus control of translational and posttranslational events that lead from mrna production to secretion . Other investigators have demonstrated an increase in vegf gene expression and secretion [1012] and a decrease in the secretion of mcp-1 by msc in hypoxia . Our results generally did not demonstrate an increase in vegf after hypoxia, but this factor also responded differently to serum reduction in our hands (see below). In addition, hypoxia increased gene expression for placental growth factor, heparin - binding epidermal growth factor, mmp-9, and basic fibroblast growth factor (bfgf) [10, 12], caused an increase in the secretion of transforming growth factor-2, insulin - like growth factor binding proteins 2, 3, 4, and 6, insulin - like growth factor- (igf-) ii, and interleukin-7 [11, 15], and reduced the secretion of stromal cell derived factor-1, macrophage colony stimulating factor, interleukin-1ra, rantes, cxcl1, and cxcl10 by msc . Clearly, hypoxia changes the paracrine secretions of msc which would have implications for the role they play in maintaining myocardial function after coronary artery occlusion . Principle among these is the stabilization of hypoxia - inducible factor- (hif-) 1 in an oxygen dependent manner between 0.5 and 5% which may be due to the release of reactive oxygen species by the mitochondria that prevents its degradation . This raises the concentration of constitutively expressed hif-1 which when combined with hif-1 forms an active transcription factor promoting cell survival . A variety of genes are affected by this cascade including chemokine receptors and those that promote anaerobic respiration, reduce aerobic respiration, and are involved in notch signaling . Hif-1 mrna expression was elevated in adipose - derived msc after 48 hours of culture in the presence of 1% o2 . In light of this, it is not surprising that we found that hypoxia alters the gene expression and secretion of paracrine factors, and hif- may be involved in the response . This study demonstrated that reducing the serum concentration from 20% to 5% caused an increase in the mrna expression for mmp-2 but a significant decrease in the expression for vegf (normoxia only), mip-1 (hypoxia only), and mip-1 (hypoxia only) while mcp-1 was unaffected . Secretion of mmp-2 and secretion of mip-1 were both significantly elevated by serum reduction while vegf and mcp-1 were significantly reduced and mip-1 was unaffected . Alterations in proliferation, gene expression, and secretion after serum reduction are not surprising since the media contain less protein and fewer signaling growth factors . Other studies have demonstrated that serum reduction (to 2%) or deprivation causes an increased expression and secretion of vegf by msc [20, 22, 23]. It is unclear why lowering serum reduced expression and secretion of vegf in our study while others found it to be elevated, but this could be attributed to our use of serum that contained stimulatory supplements (mesencult; stem cell technology) instead of fbs . However, we have previously demonstrated that this supplement does not contain vegf, mip-1, mip-1, or mcp-1 which was confirmed by the manufacturer, so that the changes observed are not simply due to a reduction in endogenous vegf . Reports from other laboratories have shown that reducing serum from 20% to 2% in msc cultures resulted in a significant decline in proliferation, an increase in the secretion of hepatocyte growth factor, and an increased expression of genes for placental growth factor, angiogenin-1, and bfgf . Serum deprivation led to an increase in the gene expression and secretion of igf-1, leptin, and angiogenin [23, 28] by msc . Since coronary artery occlusion results in a serum - deprived state in the myocardium, these results have significant therapeutic implications for the paracrine actions of msc . Hypoxia increased the expression of the pluripotent genes oct-4 and nanog in msc and stimulated the expression of a group of genes that maintained their undifferentiated state . Hypoxia promoted osteogenic and chondrogenic differentiation of msc but reduced adipogenic differentiation of msc [15, 16] and promoted development of the intervertebral disc phenotype . Reduction of serum levels to 2% caused an increased expression of stemness genes (atp binding cassette g-2, bone marrow stromal cell antigen-1, fgf-4, frizzled-9, sox-2, and oct-4), angiogenic genes (placental growth factor, vegf, angiogenin-1, and bfgf), and endogenic genes (cd34, vascular endothelial cadherin, endothelial cell adhesion molecule-1, von wildebrand factor, and vegf receptor-2) in msc . Serum reduction led to increased cardiomyocyte and endothelial differentiation [21, 23] by msc . Therefore, the hypoxic / low serum conditions present after coronary artery occlusion provide conditions that maintain the pluripotency of msc while also promoting their ability to differentiate . This change in the differentiation state might also affect the expression and secretion of paracrine factors . The paracrine factors secreted by msc have significant biologic activities that are believed to play a role in their cardioprotective effects . We have demonstrated that msc conditioned media (cm) promote angiogenesis in canine vascular endothelial cells, mcp-1 or msc - cm reduced caspase-3 activity in h9c2 cells, and mcp-1 and mip-1 promoted msc migration and vegf inhibited msc migration . Vegf is a well - known promoter and regulator of angiogenesis, and mcp-1 has proangiogenic effects as well [34, 35]. Mcp-1, mip-1, and mip-1 [37, 38] play a role in the modulation of immune function, and immune modulation may be an important mechanism whereby msc aid tissues in recovery from injury including myocardial infarction . Mmp-2 is a gelatinase that digests collagen and is tightly regulated by a variety of factors including the tissue inhibitors of metalloproteinases which are also secreted by msc . The secretion of mmp-2 by msc that we and others have demonstrated may be important in the transendothelial and tissue migration of msc [24, 43] and in angiogenesis . Differential regulation of these paracrine factors by hypoxic and low serum conditions as demonstrated in this study may be important in the beneficial effects of msc injection during the ischemia present following coronary artery occlusion.
Multiple randomized studies have demonstrated equivalent survival outcomes with mastectomy versus breast conservation therapy (bct, breast conservation surgery (bcs), and adjuvant radiation therapy) in the treatment of early stage breast cancer [1, 2]. In addition, the oxford meta - analysis convincingly demonstrated not only a significant local control benefit but also an overall survival benefit with adjuvant breast radiation therapy after bcs . As such, bct has been established as the standard of care for limited stage breast cancer offering the advantage of breast preservation, improved quality of life, and cosmesis . Breast conservation surgery, except in rare cases, is followed by adjuvant radiation therapy (rt). A typical adjuvant radiation course is 45 to 50 gy in 25 to 28 fractions (1.8 to 2 gy per fraction) delivered to the whole affected breast . A boost of 1016 gy in 5 to 8 fractions is usually prescribed to the lumpectomy cavity for additional local control benefit as demonstrated by two seminal studies [4, 5]. With the standard rt schedule, a 57 weeks commitment is required . For most patients, this is quite inconvenient and cumbersome because of employment or social responsibilities, often confounded by remote distance from the treatment center to their place of work or residence . In fact, this treatment time commitment has been cited as one of the main reasons for noncompliance with adjuvant breast radiation . This demand served as impetus for the development of abbreviated regimes for whole breast radiation . One such schedule is the widely adopted canadian fractionation, 42.5 gy in 16 fractions (2.65 gy per fraction) given over 3 weeks . This and other regimens, 40 gy in 15 fractions and 39 gy in 13 fractions delivered over 3 weeks, partly borne from its inception, are discussed in detail herein [711]. Conceptually, a shorter course of radiation therapy necessitates hypofractionation utilizing a higher dose per fraction to achieve radiobiological equivalent effectiveness of the standard, more protracted schedule . From radiobiological principles, since late reacting normal tissues are more sensitive to increasing dose per fraction, a priori, larger dose per fraction should yield more long - term toxicity . Thus, the tenable, reserved position among some physicians is that the abridged regimens may be tagged with the high clinical price of long - term treatment toxicities . The utilization of hypofractionated whole breast rt has been contemporaneous with a more focused approach, accelerated partial breast radiation therapy (apbi). As the name implies, radiation is targeted to the partial breast only, defined by the lumpectomy cavity borders and up to a 2 cm margin diametrically . Accelerated hypofractionated course of radiation, with a schedule ranging from 20 gy administered in 1 fraction as in the case of intraoperative radiation therapy (iort) to 38.5 gy in 3.85 gy fractions twice daily over 5 consecutive days with external beam radiation . The underlying principle of partial breast irradiation is that over 85% of all ipsilateral breast recurrences occur in the same quadrant within a 1 - 2 cm radius of the index lesion . By this premise, partial breast rt should not significantly compromise treatment outcomes compared to whole breast radiation, in low - risk patients . In addition, organs at risk (oar) for radiation - induced toxicity such as the unaffected contralateral breast, lungs and heart should be less threatened by partial breast than with whole breast radiation therapy . Partial breast radiation offers a clinically desirable constellation of advantages over conventional radiation, including shortened treatment duration and reduction of normal tissue toxicity . This begs the question: can we confidently minimize target volume without compromising treatment outcomes? Image guidance promises to provide improved accuracy in target localization that should allow target volume reduction . Herein, we review the results from the most relevant hypofractionated whole and partial breast studies and discuss the implications thereof . We conclude with a brief discussion of image guidance and its utility in whole and partial breast radiation . Several randomized clinical trials have compared the efficacy of whole breast radiotherapy with conventional fractionation (i.e., 1.82.0 gy fractions) requiring five to six weeks of daily treatments versus hypofractionated (i.e.,> 2 gy fractions) radiotherapy requiring fewer treatments . Overall, these trials have shown equivalent local control of breast cancer and breast cosmesis with conventionally fractionated versus hypofractionated regimens . A canadian trial compared hypofractionated whole breast regimen delivering a dose of 42.5 gy in 16 fractions of 2.66 gy daily fractions over 22 days to conventional fractionation of 50 gy in 25 daily fractions of 2 gy each . Both regimens were prescribed without a sequential lumpectomy cavity boost . With median followup of 12 years, the 10-year local control was 93.3% versus 93.8% (p> 0.05) for the conventional versus hypofractionated radiation, respectively, with both regimens yielding equivalent cosmesis . Another trial centered at the royal marsden hospital compared conventional 50 gy in 2 gy fractions to two hypofractionated arms, delivering 39 gy or 42.9 gy in thirteen 3.0 gy or 3.3 gy fractions, respectively, over 35 days . That study yielded equivalent 10-year local control rates of 87.9%, 85.2%, and 90.4% for conventionally fractionated and the two hypofractionated radiotherapy regimens, respectively . Two additional uk trials, start a and start b (standardization of breast radiotherapy), with shorter followup (5-year) similarly demonstrated equivalent local control between treatment arms [16, 17]. In contrast, an ongoing uk fast trial (faster radiotherapy for breast cancer radiotherapy) randomized patients between conventionally fractionated radiotherapy and two hypofractionated schedules, 28.5 gy in five 5.7 gy fractions and 30 gy in five 6 gy fractions delivered over 35 days . The results of this trial have not yet been reported in full - text form [18, 19]. The question of feasibility of delivering lumpectomy cavity boost after canadian and other fractionated whole breast schedules has been posed . Both start and the royal marsden trials prescribed a boost in more than 30% of the study cohort [1517]. This has also been addressed by a single institution memorial sloan kettering cancer center retrospective series in which hypofractionated whole breast radiation therapy (42.4 gy in 16 fractions of 2.65 gy each) was delivered to 128 patients followed by a conventionally fractionated boost of (10 gy in 5 fractions of 2 gy each). That study showed comparable cosmetic outcomes to conventional fraction, and there were no grade 3 or more toxicities recorded after median followup of 1.5 years . Another large single institution uk series confirmed feasibility and favorable outcomes with canadian fraction followed by boost . In 2010, the american society for radiation oncology (astro) published evidence - based guidelines for hypofractionated whole breast radiotherapy . Suitable candidates for hypofractionated radiotherapy are identified as women aged 50 years with pt1 - 2 n0 m0 tumors and who do not receive cytotoxic chemotherapy . With the latter criterion it is still uncertain whether all women benefit equally from hypofractionated as compared to conventionally fractionated radiotherapy regimens . A retrospective exploratory subgroup analysis of the canadian trial revealed that the hypofractionated regimen was less effective among women with high - grade tumors (10-year local recurrence 15.6% versus 4.7%, hypofractionated versus conventional fractionation regimens, resp . ). Additional data and continued followup from randomized trials will be important in determining the long - term efficacy and cosmesis from hypofractionated whole breast radiotherapy regimens . Accelerated partial breast irradiation (apbi) is another technique used to deliver a course of radiotherapy over an even shorter time frame of, usually, 5 days . This volume can be targeted with various radiotherapy techniques such as external beam radiotherapy (either 3-d conformal, intensity modulated, or electron radiation therapy), brachytherapy (interstitial or balloon catheter), and intraoperative radiotherapy (electrons or superficial photons). Several randomized clinical trials comparing whole breast to accelerated partial breast irradiation are ongoing . The nsabp b-39/rtog 0413 trial, goal accrual of 4300, randomizes patients between whole breast irradiation or accelerated partial breast irradiation (with choice of either high - dose rate interstitial brachytherapy, mammosite balloon catheter, or 3d conformal external bream radiotherapy technique) to a dose of 34 gy or 38.5 gy in 3.4 gy or 3.85 gy fractions over 510 days . Final results of this trial have not yet been reported . The largest reported apbi trial to date, targit (targeted intraoperative radiotherapy) trial, randomized 2232 women (excluding patients with certain high - risk clinicopathologic features) between whole breast irradiation and a single dose of 20 gy with intraoperative radiotherapy with superficial low - energy photons . At 4 years, there was no difference in local control between the whole breast (99.1%) and partial breast (98.8%) arms . Results employing electron intraoperative therapy (eliot) in 1822 women with early stage breast cancer have been published, demonstrating 97.7% local recurrence rate at 3 years and a 5- and ten - year survivals of 97.4 and 89.7%, respectively, while offering reduction of normal tissues to radiation exposure . European society for therapeutic radiology and oncology); rapid (randomized trial of accelerated partial breast irradiation); import low (intensity modulated and partial organ radiotherapy) are examples of current randomized trials evaluating accelerated partial breast radiotherapy versus whole breast irradiation in the treatment of low - risk breast cancer . The treatment and cosmetic outcomes of mature and ongoing clinical trials should help to clarify treatment criteria and appropriately stratify patients to partial breast versus whole breast irradiation . Image guided radiotherapy (igrt) involves the use of localization techniques at the time of daily treatment to verify accurate positioning . The goal of this endeavor is to reduce patient setup variation, in order to facilitate the use of smaller margins around target volumes to be used . Smaller margins should translate into significantly smaller volume of normal tissue radiated which should in turn reduce acute and late normal tissue toxicity . In the case of breast cancer treatment, this approach may reduce late effects to the breast tissue, heart, and lungs . Used in conjunction with localization of the target volume, particularly in the case of partial breast irradiation, igrt may additively improve daily target dose coverage and therefore improve local control outcomes as well . Even in the setting of whole breast treatment, the use of igrt may facilitate smaller margins as well as advanced techniques such as simultaneous integrated boost and intensity - modulated radiation therapy (imrt), both of which require a much higher degree of setup certainty to be effectively used in treatment [26, 27]. Aligning to bony anatomy, as is done in mv and kv or cone beam ct imaging, improves setup accuracy compared to the traditionally employed surface tattoos [28, 29]. Using either breast surface or location of intraparenchymal surgical clips improves localization over strictly bony alignment, even when using ct guidance . Still, there is some uncertainty associated with the delineation of the tumor bed target volume because of significant interobserver variability [31, 32]. For example, the radiation therapy oncology group (rtog) conducted a multi - institutional interobserver study among nine radiation oncologists specializing in breast cancer, to determine the degree of variability in target volume and organs at risk delineation among three sample cases . They found structure mean overlap of only 72% for the lumpectomy cavity in one case and poor agreement on nodal structures, with percent overlap as low as 10% among different observers . Consequently, the rtog has published an atlas of breast, chest wall, nodal regions, and organs at risk to guide a more consistent reproducible approach to contouring . We have recently reported our experience at moffitt cancer center using fiducial - based igrt in prospective cohort of both whole breast and partial breast patients . We used textured gold fiducial markers, which adheres to the surrounding soft tissue, increasing the likelihood of fiducial stability and consistent visualization . In fact, 100% fiducial visualization on mv imaging and minimal variation, we were able to verify fiducial migration . In the partial breast cohort, there was minimal motion due to intrafraction motion from respiration or changes in respiratory motion between 4d ct scans at the time of simulation to the end of treatment . The mean change in distance between fiducials inter- and intrafraction had a small range 2 to 3 mm, well within the range of error of the total size of the fiducial . Fiducial markers position was stable during treatment with no evidence of substantial fiducial migration within a 5 mm range . The position of the center of fiducial mass relative to the center of the seroma was also stable, confirming the stability and applicability and textured fiducials in igrt in the setting of apbi . Similarly, in the whole breast cohort, small ranges in inter- and intrafraction motion, respiratory motion, and fiducial migration were observed . Our data suggest that, with fiducial - based image guidance, the ptv margin may be safely reduced from the more standard 10 mm to about 5 mm, substantially reducing the volume of normal tissue irradiated unnecessarily . Other investigators using surgical clips or fiducials for breast cancer radiotherapy have reported similar results [3741]. Although online cone beam ct (cbct) allows much more accurate and reproducible alignment in 3 dimensions to the bony anatomy as compared to surface tattoos or port films alignment, pretreatment cbct does not guarantee accurate intrafraction delivery [42, 43]. Prolonged treatment times and couch rotation can significantly reduce treatment delivery accuracy . To address this confounder, patient surface setup systems and real time tracking systems data suggest that surface imaging may offer more precise setup than laser or tattoo with a similar reduction in error as fiducial - based igrt . Ultrasound systems for localization and tracking of the tumor bed for daily treatment have also been investigated and have shown good correlation between the position of the tumor bed on 3d ultrasound and ct, forecasting the utility of 3d us in the near future . Similarly, implantable electromagnetic transponder fiducials have been used to track breast tumor bed motion in real time . With the rising concern of cumulative radiation doses with multiple ct imaging and the lifetime risk of secondary cancers, modalities such as hypofractionated breast radiation therapy offers the attractive alternative of shorter treatment course which is not only convenient for patients, but also time and cost - effective . The overarching question remains, can we minimize without compromise? Results of randomized and large single - institution studies seem to support the edict that attaining this desirable balance is indeed possible . Reducing target volume, as in the case of external beam apbi techniques, calls for more refinement of treatment delivery with image - guided radiation therapy to ensure accurate delivery of high - dose radiation while sparing normal tissue such as heart and lungs . Various methods of performing igrt, including implanted fiducials, cbct, surface mapping, and ultrasound afford measurable improvement in setup error allowing for ptv margin reductions to as much as 5 mm . Each institution should apply the optimal igrt technology for their clinical practice commensurate with the center's equipment availability, physician, and technician experience . For, whole breast radiation, wherein a larger volume of heart and lung is irradiated, there is a compelling argument to incorporate igrt in our daily set up, to achieve optimal results with minimal toxicity . For accelerated partial breast irradiation in particular,
Surveys on infection by schistosoma mansoni that have been conducted in minas gerais since the 1970 decade have aimed to follow up the natural history of schistosomiasis with emphasis on (1) the pathogenicity of the etiological agent and its experimental behavior, (2) the host's characteristics and the influence of race, occupation, education level, age group, sex, specific treatment and frequency of contact with water streams for the activities of washing clothes, leisure, and fishing [2, 3], and (3) environmental conditions, distribution of streams and their distance to homes, presence of biomphalaria, and its infection rates and infectivity, with comparison of exposure to different isolates of the parasite [4, 5]. The morbidity of infection has been evaluated through clinical and epidemiological studies in two field areas, in the rio doce and jequitinhonha valleys of the state of minas gerais, brazil . Patients with hepatosplenic forms have been evaluated at the university hospital of the federal university of rio de janeiro, brazil . The aim of the present work was to compare the prevalence of infection by s. mansoni in these two areas, the morbidity due to schistosomiasis according to classification of clinical forms of the disease . The present survey was carried out in two rural areas of minas gerais: area 1so joo, and area 2municipality of capito andrade, located, respectively, in the jequitinhonha and rio doce valleys, at distances of 890 km and 600 km from the state of rio de janeiro . The total number of inhabitants taking part in the survey was evaluated according to a random number table . In area 1, 75 homes were sampled, involving 288 individuals; and in area 2, 80 homes with 257 individuals . Stool parasitological tests were based on the kato method, as modified by katz et al ., and the intensity of infection was evaluated using the median number of eggs per gram of feces, according to the inhabitants' ages and gender . In order to measure liver and spleen sizes, the subjects were examined in the dorsal position (during deep expiration) and in the schuster position . Ultrasonography was requested to determine the portal vein caliber, liver and spleen sizes, and fibrosis . This study was approved by the ethical committee of the instituto de pesquisas clnicas evandro chagas - fiocruz (conep protocol 0127.0.011.009 - 05). The clinical classifications on both populations were based thus in three clinical forms: type i schistosomiasis infection, with or without symptoms, which, if present, were moderate and not necessarily attributed to the disease; type ii hepatointestinal form, with frequent intestinal symptoms, such as dysenteric diarrhea and hepatomegaly; and type iii hepatosplenic form, with very frequent intestinal symptoms of dysentery and hepatosplenomegaly, with or without hematemesis or melena [8, 9]. During the clinical examinations on this population, the subjects were asked about intestinal symptoms of dysentery, hematemesis, and/or melena . Those with hepatosplenomegaly were admitted to the clementino fraga filho university hospital in rio de janeiro and were followed up by the teams of the infectious and parasitic diseases service and the surgical sector . Demarcated sites of water streams were examined for biomphalaria, with the aims of classifying the mollusk species that were present and determining the cercarial infection rate, after exposure to light . If the biomphalaria specimens were negative, they were infected experimentally with s. mansoni isolates from patients from different regions of brazil, to test the mollusks' susceptibility to this infection . The statistical analysis was based on the chi - square test with a confidence level of 95% (p <0.05). A total of 288 parasitological stool tests were carried out on individuals in the comunidade so joo, comprising 163 females and 125 males . The prevalence rate of s. mansoni infection was 22.9% (33.6% in males and 14.3% in females). Men were twice as likely to acquire infection as women were (relative risk = 2.2), with the highest rate in the age group between 11 and 30 years old . There were 257 examinations on individuals in the population of capito andrade (108 in males and 149 in females). The prevalence of infection was 20.2% (30.5% in males and 12.7% in females), predominantly in men (relative risk = 2.4) and in the age group between 11 and 30 years old . The number of s. mansoni eggs per gram of feces ranged from 24 to 48 in both areas, showing that the infection presented low intensity . In the so joo community, 69.2% of the infected individuals presented the schistosomiasis infection form; 28.7% presented the hepatointestinal form; and 2.1% presented the hepatosplenic form . Two cases (one is 12 years old and the other is 13 years old) had the diagnosis of schistosomal myeloradiculopathy, for which the main symptoms were headache, limb and back pain, weakness, paresthesia, and paraplegia . These two patients underwent neurological examination and the venereal disease research laboratory, hiv antibody, cytomegalovirus, and herpes virus tests . Magnetic resonance imaging revealed dilatation of the medullary conus, and the likely source was indicated as schistosomal myelopathy . After treatment with a single oral dose of praziquantel (40 mg / kg), the symptoms regressed, with no sequelae over a three - year follow - up period . In capito andrade, 70.2% of a total of 65 positive inhabitants of the studied sample were classified as presenting the schistosomiasis infection form; 26.5% presenting the hepatointestinal form; and 3.3% presenting the hepatosplenic form (table 1). The presence of esophageal varices was not confirmed through digestive endoscopy in the cases of the individuals with hepatosplenomegaly . No cases of ascites, cirrhosis, or other complications indicating liver involvement caused by comorbidity associated to s. mansoni infection was observed . None of them were found to be positive for cercarial elimination during the period of the study, but they were seen to be susceptible when infected by s. mansoni isolates of patients from different regions of brazil . This disease affects at least 240 million people around the world, and more than 700 million people live in endemic areas . Moreover, the infection is prominent in nineteen brazilian states, especially in minas gerais, which accounts for about 70% of the endemic areas for the disease [12, 13]. The longitudinal surveys started in minas gerais emphasized that the morbidity and prevalence rates decrease when a specific treatment of infected individuals is associated with improvements of sanitary conditions . Different authors in brazil have contributed towards studies on the influence of treatment on prevention of severe clinical forms of this disease [1519]. There was no difference in the prevalence of infection by s. mansoni between so joo (22.9%) and capito andrade (20.2%). Both areas highlighted a gradual increase in the rate up to the age of 11 years, with a decrease after the age of 50 years . The prevalence of rates higher than 20.0% in both areas indicates that these areas are included in category ii with moderate prevalence (20% and <50%). There was higher prevalence among males than that among females, such that men were twice as likely to become infected as women were . The intensity of infection was low, ranging from 24 to 48 eggs per gram of feces . It was more precarious and poor in quality in so joo, where the current study began . No comparisons can be made with any previous surveys in this area, although two cases of schistosomal myeloradiculopathy can be highlighted . These showed regression of symptoms, without sequelae, after treatment with praziquantel and monitoring of case evolution for three years after the infection had been treated . There was no difference in the prevalence of infection by s. mansoni between so joo and capito andrade, with a low intensity of infection and a higher rate among males up to the age of 11 years . It was highlighted two cases of myeloradiculopathy in the jequitinhonha valley, with regression of symptoms after specific treatment . The factors determining this lack of control may include low sensitivity and the small number of diagnostic procedures performed, as well as no response to the drugs used . Other additional factors that might explain the maintenance of transmission rates include the poor sanitation system, the lack of early specific treatment, and the reinfection processes.
The genetic composition of a natural population is always changing because mutations inevitably arise and rise or fall in frequency due to selection or drift (i.e. Stochasticity in finite populations). Genetic changes continue during invasions, when a population is entering and becoming established in new territory . Ecologists recognize that selection can play a crucial role in determining the success, speed and ultimate extent of invasions (hoffmann and sgr 1995; sakai et al . Even neutral genetic change during invasions is of high interest to researchers who use neutral markers to infer the migration history of populations (semino et al . 2000; sunnucks 2000; schaal and leverich 2001; wang and whitlock 2003; kawiecki and ebert 2004). This is because to test any hypothesis concerning the past spatial distribution of a population, it is necessary to specify a null model that incorporates explicit assumptions about demography and dispersal patterns . It is further necessary to understand the distribution of neutral allele frequencies under the specified model, in order to compare observed marker data with the distribution expected under the null hypothesis . Yet, despite the recognized importance of genetic changes during invasions, theoreticians have paid little attention to this topic until recently (work on the evolution of dispersal constitutes an important exception; see, for example, holt 2003; simmons and thomas 2004). Recently, several authors have used stochastic simulations to study the fate of a single mutant that arises during an invasion (edmonds et al . Mutation surfing, in which mutants arise on or advance to the front of a traveling wave of population density and essentially shut out wild types (i.e. Nonmutants) from a certain point in the invasion onwards (edmonds et al . Early studies of this topic offered a number of descriptive statements about the fate of mutations in invasions, including a midpoint rule for the location of the center of population density of a successful neutral mutation (edmonds et al . 2004) and a proposal that a certain lumped model parameter has a strong linear correlation with the ultimate fraction of the invading population that carries such a mutation (klopfstein et al . 2005). More recent studies have treated surfing with selection, using mathematical models such as annihilating random walks, generalized diffusion equations and stochastic partial differential equations to explain the phenomena observed in surfing simulations and in vitro experiments (hallatschek et al . 2007; hallatschek and nelson 2009) (korolev et al ., 2010). Recently, it has been argued that some genetic differences between human populations that had previously been attributed to selection in fact resulted from surfing by neutral alleles (hofer et al . One purpose of the present work is to provide a careful qualitative and quantitative description of neutral mutation surfing as seen in a stochastic model like those studied in edmonds et al . We focus on a model of a one - dimensional habitat but include some results for two - dimensional habitats . It should be noted that a one - dimensional habitat is a realistic model for certain types of invasions, such as invasion along a coastline or river (lubina and levin 1988; speirs and gurney 2001; pachepsky et al . 2005). As a consequence, study of such models and comparisons between them and first, neutrality is simpler than selection, and with so little existing theory, it is reasonable to study the simpler case first . Second, neutral genetic markers are of interest because they can provide information about the history of an invasion . Indeed, much of the original interest in mutation surfing was among researchers whose primary goal is to reconstruct range expansion (e.g. The spread of humans into europe) with such markers (edmonds et al . 2004). On the other hand, adaptive change during invasions may be the genetic phenomenon of most practical interest to conservation biologists . Accordingly, we have begun to extend our models to the cases of beneficial and deleterious mutations, and we present some results here . Our main goals are to describe how the probability of surfing depends on model parameters (with or without selection), to explain heuristically the nature of this dependence, and to offer a simple model of the surfing process as a contribution to the development of analytic models that yield quantitative predictions about genetic change during invasions . Our work is based on data obtained from a series of simulations of cellular automata . In what follows, we state the specifications of the simulations, use statistical methods (in particular, logistic regression) to describe the probability of surfing and to assess our analytic model, and offer likely explanations for the quantitative results we obtain . The model we studied, following edmonds et al . (2004), klopfstein et al . (2007), is a type of individual - based model known to mathematicians as a contact process (liggett 1999). We simulated a contact process in which wild - type (i.e. Nonmutant) and mutant individuals reproduce asexually and move between adjacent cells in a rectangular grid . For the neutral case, grid lengths l used were 100, 200 and 400 cells; grid widths w used were 1, 3, 7, 13 and 25 cells . For the case of selection, only 1400 grids were used . We note that previous studies used only 25 100 grids (edmonds et al . We varied grid width in order to study the effect of dimensionality on the probability of surfing . We varied grid length in order to make sure that numerically ascertained probabilities of surfing on a finite grid came close to asymptotes, which we expect to correspond to probabilities of surfing on an infinitely long grid . Accordingly, all results below pertain to grids of length 400 unless otherwise specified . As in klopfstein (2005), each simulation run began with a single wild - type individual placed at the center of the leftmost column of the grid . For example, the founder could be placed along a side or in the middle of the grid to model colonization beginning other that at the mouth of a river . First, each individual was replaced in the same cell by a number of offspring chosen from a poisson distribution with mean . For the neutral case, we used 1 = 0.05, 0.1, 0.2, 0.4, 0.8, 7, 15 and 31; in each simulation run, was the same for mutants and wild types . For the case with selection, we used wt 1 = 0.1 and 0.4 for wild types and mut 1 = (wt 1) for mutants, with = 0.5, 0.95, 1.05 and 1.5 . Next, each cell whose population n (of mutant and wild types combined) was greater than a carrying capacity k underwent culling, in which nk randomly chosen individuals were discarded . All individuals within a cell, whether mutant or wild type, had equal probability of being culled . For the neutral case with <2, we used carrying capacities k = 10, 50, 100 and 500; we used k = (1)/2 for all> 2 . For the case with selection each individual (whether mutant or wild type) had a probability m of migrating in each generation . For the neutral case with <2, we used mean migration rates m = 0.05, 0.1, 0.2 and 0.4; with> 2, we used m = 0.1 and 0.2 . For the case with selection, we used m = 0.05 and 0.2 . Each individual chosen to migrate then moved to a cell chosen with equal probability from all those adjacent to its original cell . (cells in the interior of the grid had four neighbors, those on the boundary had three and those in the corners had two; diagonal moves were not permitted). Predictors, estimated coefficients with standard errors and 95% confidence interval lower and upper bounds for odds ratios for one - dimensional habitat with k = 10 when at least one member of the population reached the 10th column from the left, one (wild - type) individual chosen randomly with equal probability from all individuals in the 10th column was replaced by a mutant . Thus, each offspring shared the type of its parent, with the sole exception being the original mutant . A run was stopped after the first generation in which either the entire population went extinct, mutants went extinct after being introduced or at least one individual reached the rightmost column of the grid . After a run was stopped, a code for the state of the mutant and wild - type populations was saved . Three states were distinguished for each type: extinction (no individuals of a given type persisted), persistence without advancing (at least one individual remained, but no individuals of the specified type were found in the rightmost column) and advancing (at least one individual of the specified type was found in the rightmost column). Experimentation showed that wild types virtually never went extinct after a mutation event had occurred; so, runs with mutation events were counted as successful, even though they were usually aborted if the mutants subsequently went extinct . (as the total population size was close to 10k times the habitat width when the mutant was introduced, it is not surprising that extinction almost never occurred after this time .) Surfing was considered to occur in runs where a mutant advanced to the rightmost column . When this occurred, it was extremely rare for wild types to also be found in the rightmost column . In addition to outcome codes for each run, we saved the state of the 7h subgrid centered on the 10th column for the three generations immediately following introduction of the mutant . For the neutral case with a one - dimensional (1 400) grid, each possible combination of the model parameters, k and m was used in 600 simulation runs . For the case of selection, each combination of the model parameters was used in 10 000 runs . For each two - dimensional (w 400 with w = 3, 7, 13 and 25) grid, each parameter was used in 50 simulation runs . We also performed small numbers of runs with parameter combinations other than those listed here; specifications for these runs are given when they are discussed below . Unlike in klopfstein et al . (2005), sequences of runs were not stopped when a predetermined number of instances of surfing was reached . This avoided bias in estimating the probability of surfing, as otherwise a negative binomial distribution would have had to be used to model this probability (hogg and tanis 1997). Visual inspection of runs in progress gave a strong impression of a dichotomy of outcomes: mutants either vanished or flourished (see figs 1 and 2). In many runs, mutants went extinct . In most runs (and in nearly all runs on grids of length 400) where mutants persisted without advancing, only a few mutants remained on the grid at the end of the simulation, and these few were found close to their point of origin . Longer habitat lengths led to more mutant extinctions and fewer cases of mutant persistence without advancing . This was apparently because runs on longer grids simply took longer, allowing more time for nonsurfing mutant populations to dwindle and ultimately die out . Wild - type (circle) and mutant (star) population densities as functions of longitude on a 1 400 grid after 20, 40, 80 and 300 generations in a run with mutant surfing . Wild - type (circle) and mutant (star) population densities as functions of longitude on a 1 400 grid after 20, 40, 80 and 300 generations in a run with mutant extinction . Parameters are as in figure 2 . Visual inspection also showed that in virtually all runs where mutant surfing occurred, the mutants excluded wild types from the wavefront very soon after the initial mutation arose, and proceeded to nearly monopolize the habitat between the point of mutant introduction and the right - hand boundary . We thus viewed all successful runs as random trials with two possible outcomes, surfing or mutant extinction . For a given combination of parameters, we denote the probability of surfing (conditioned on nonextinction of the total population) by psurf . We note that under neutrality, the left - hand boundary of the habitat occupied by surfing mutants remained close to the point at which the mutation first arose . Surf subsequently lost habitat as wild types advanced to replace the less - fit types . A major goal of work on mutation surfing is to relate the probability of surfing to model parameters such as cell carrying capacity k, migration rate m and growth rate (edmonds et al . 2004; klopfstein et al . 2005; hallatschek and nelson 2008). For the one - dimensional case, plots of mean (over all runs with given values of, m and k) psurf against model parameters showed an interaction between k and (fig . 3). For each value of k, psurf increased with . However, the rate of increase was much greater for the smallest carrying capacity, k = 10, than for the higher values of k. this motivated dividing the runs into two groups, one with k = 10 and one with k = 50, 100, 500, for further analysis . Ln (psurf) versus ln (1) for a one - dimensional habitat, with regression lines for k = 10 (circles) and 100 (squares). To quantify the dependence of psurf on k, m and, we use logistic regression, a tool which is adapted to processes with binary outcomes for individual trials (hogg and tanis 1997). In our logistic regression, the response (or y) variable is the log - odds of surfing: the predictor (or x) variables could in principle be any or all of the model or simulation parameters, or functions of those parameters . In seeking an appropriate statistical model to describe the simulation output, we tried various functions of k, m and as predictor variables . We used three different goodness - of - fit tests (pearson's chi - squared, deviance and hosmer lemeshow) (hosmer and lemeshow 2000) to assess the suitability of each statistical model . We used three criteria to choose among the models that passed the goodness - of - fit tests at the 1% significance level . First, we sought a model in which every predictor was associated with an odds ratio that differed significantly from 1 . Third, we sought a model with a low value of the akaike information criterion (aic) (akaike 1974). We judged the most suitable model to be a function of ln m, ln (1) and ln k: (standard errors for the coefficients and confidence intervals for odds ratios are given in table 2). We used mcfadden's pseudo - r (r) to assess the amount of variability explained by equation (2); we found r = 0.31, indicating reasonably strong explanatory power . Predictors, estimated coefficients with standard errors and 95% confidence interval lower and upper bounds for odds ratios for one - dimensional habitat with k = 10, 100, 500 for the same one - dimensional grid with k = 10, we did not find a satisfactory model . This may have been because values of psurf with k = 10 were typically very low, so that the relative sampling error in estimating psurf was high enough to obscure associations with the other model parameters; further simulations are planned to explore this possibility . It was possible to combine data for all values of k and find a model that satisfied our formal criteria, but this required including so many interaction terms that the model did not seem to be useful for gaining insights . It is possible to explain heuristically the direction of the dependence of psurf on each predictor (, m, k) in equation (2). It is well known for simpler demographic structures that the smaller the total population, the greater is the probability that a mutation initially present in a single copy will rise to high frequencies (hartl and clark 1997). Correspondingly, in our model the mutant should be more likely to surf if it initially appears in a cell with no other (wild - type) occupants . This is turn should be more likely if the cell carrying capacity k and the migration rate m are low because these conditions should lower the number of individuals at the wavefront, where the mutation occurs . Furthermore, cells at the wavefront should generally contain fewer individuals than cells further back in the wave; so, fewer offspring will be culled at the front, making the effective growth rate higher there . Again, as the mutant must arise at the front, this means that the probability of surfing should increase with (as for fixed m and k, a higher value of should allow more mutants to be born before wild types catch up to the mutant population at the leading edge). In equation (2), it is a straightforward calculus exercise to check that the derivatives and are both negative, while is positive, for the range of parameter values used . Thus, our statistical model predicts that the probability of surfing psurf will decline as m or k increases but rise as increases, which is consistent with our heuristic argument . For the (neutral) two - dimensional case, we first considered only those runs with a 25 400 grid . Using the same criteria as for the one - dimensional case, we found a simple model to provide the best description of the relationship between psurf and the model parameters: (standard errors for the three coefficients, in order, were 0.27, 0.063 and 0.13). We caution, however, that for this model r = 5% . As in the one - dimensional case, here however, including functions of the migration rate m as a predictor did not improve the model in two dimensions . Equations (not shown) with coefficients close to those in equation (3) provided good fits to simulation data with grids of widths 13 or 7; the fit was less good for a 3 400 grid . However, all results for the two - dimensional case should be viewed as preliminary because of the small number of trials performed with each combination of parameters . For the case with selection, surfing never occurred when mut was less than 1; so, we considered only parameter combinations with mut> 0 (these did include some deleterious mutations). We did not find a statistical model that simultaneously satisfied goodness - of - fit criteria and provided good explanatory power as measured by r . The model gave r=0.23 but failed goodness - of fit tests (p <0.001); standard errors for the coefficients, in order, were 0.061, 0.055, 0.030, 0.015 and .0093 . In equation (4), it is noteworthy that psurf depends on mut much more than on wt . This suggests that for the cases studied, absolute fitness was more important than relative fitness in determining surfing success (a similar effect was found in an island model analyzed by holt and gomulkiewicz (1997)). We also note that although the coefficients of ln m and ln k differ from those in the neutral case (2), their sign is the same . Thus, for the cases studied, the probability of mutant surfing varies inversely with m and k for both neutral and selected mutations . Beyond merely describing (even quantitatively) the dependence of psurf on model parameters, it is clearly important to explain the form of this dependence in a way that sheds light on the process by which a mutation succeeds or fails . A partial explanation can be obtained from two observations; we consider the neutral case first . In this case, they reproduce and migrate in identical ways, and count identically toward the carrying capacity of a cell . The second observation, based on watching generation - by - generation plots of mutant and wild - type population density as a function of longitude, is that in virtually every case when surfing occurs, mutants take the lead and begin to fill the available unoccupied terrain within a very few generations after the original mutation occurs . As mutant and wild - type individuals behave identically, we may draw a conclusion about the genealogies of individuals in the advancing wave of population density as follows . Imagine drawing a line at the longitude where the mutant first arises, or a short distance past this point . Then when a simulation run terminates, nearly all of the individuals to the right of this line will have descended from a single common ancestor in the generation when the mutant first appeared regardless of whether mutation surfing occurred . For when surfing does occur, almost all of the relevant terrain will be occupied by mutants, which must have descended from the single original mutant . Therefore, as mutants and wild types are functionally identical, similar genealogies must arise even when surfing does not occur and the terrain is filled by wild types . We can thus recharacterize mutation surfing as the scenario in which the common ancestor of nearly all individuals in the population that ultimately invades happens to be the original mutant, rather than any of the wild types present at the time the mutation occurred . (this line of reasoning informs models of the coalescent with stepping - stone spatial structure; see, for example, austerlitz et al . Our second observation that a mutation's fate appears to be sealed within a few generations of its appearance suggests a simplified model of the process by which this common ancestor is the first step occurs in a early generation (p, f1 or f2, where the generation including the original mutant is counted as p). In this step, one individual is chosen randomly from those in the rightmost cells as a potential common ancestor for the future population . Each individual in the rightmost cell, whether mutant or wild type, has the same probability of being chosen . In the second step, the chosen individual succeeds or fails to propagate with probabilities equal to the probabilities of survival or extinction at the very beginning of a run (when the population consists of exactly one individual, located at the left - hand boundary). Motivation for this step comes from the strong association between psurf and prun, the a priori probability that a run will not end in extinction, when is not very high (fig . If the chosen individual fails to propagate (i.e. Its progeny die out), the process begins again with a new choice of potential common ancestor from those which were present in the rightmost cells during the p generation (all of which are assumed to have survived). ; ln (psurf) versus ln (prun) for a one - dimensional habitat and <2 . This model leads to a testable quantitative prediction . To derive an estimate for psurf under this model, we let n denote the number of individuals present in the rightmost cell in generation p. then the first term on the right - hand side of equation (5), (1/n)prun, is the probability that the mutant is the first candidate chosen and does succeed in propagating . The second term is the probability (n 1)/n(1 prun) that a wild - type individual is the first chosen but fails to propagate, times the probability 1/(n 1)prun that the mutant is the second chosen and does propagate . The subsequent terms are derived similarly . Algebraic manipulation condenses equation (5) into the form to test whether equation (6) was a good fit to the one - dimensional simulation results, we first estimated prun and mean n separately for each combination of the parameters, m and k that yielded at least one run not ending in extinction (there were 70 such parameter combinations), then ran a linear regression of the observed values of ln (psurf) against the predicted values from equation (6). (taking logarithms rendered the variance of observed psurf approximately equal for all values of, a necessary condition for valid inference in linear regression .) We caution that in our simulations when was very high (i.e. 8), prun was always very close to 1 and so a relationship between prun and psurf was no longer apparent; therefore, the following discussion considers only our neutral simulations with <2 . The regression equation was (r=56.1%), which is broadly consistent with prediction (6), except that the constant was lower than expected (standard errors for the constant and the coefficient of ln (prun) were 0.1766 and 0.1120 respectively). When the regression was run for each value of the migration rate m separately, the discrepancy between predicted and observed psurf was seen to occur only for very high migration rates . Indeed, the constant term and the coefficient of in the regression equation were not significantly different from the predicted values of 0 and 1, respectively, except for the highest value of m, namely 0.40 . Agreement between the predicted and observed equations improved as m decreased (details are shown in table 3). Coefficients for regression of observed on predicted ln (psurf) we now briefly consider model (6) for large . When and k are both very large, prun approaches 1, and taking a limit of the right - hand side of equation (6) with k being a fixed multiple of 1 (k = a(1)) and prun = 1 e suggests that psurf should approximately equal 1/. However, our simulations with large and k = (1)/2 show far higher values of psurf than this calculation would predict for example, psurf 0.85 with = 32, k = 16 and m = 0.1 . The explanation appears to be that even though most cells filled to capacity within one or two generations of colonization, the rightmost cell generally contained very few individuals averaging fewer than two for all conditions simulated and each of these individuals had a high probability of becoming the common ancestor of the future occupants of the grid from that point onwards . (it is possible that different behavior might be found if k were held constant as increased, however .) Analogues of equation (6) proved to be almost completely unexplanatory in two - dimensional settings under neutrality, yielding r values of less than 5% . Indeed, there appeared to be no meaningful relationship between any powers of psurf and prun in two dimensions . Under selection (i.e. With mut wt), the analogue of equation (6) is more complicated because the numbers of mutant and wild - type candidates remaining after each unsuccessful attempt to propagate do not follow a simple linear pattern . Rather, if the rightmost cell in generation p is occupied by one mutant and n 1 wild types, and if the number of candidates is still assumed to decrease by 1 after each failed attempt, then the expected numbers u(t) and w(t) of mutant and wild - type candidates t generations later (conditioned on mutant survival) are given by when these expressions are used in an analogue of equation (6) that also includes different values of prun for mutants and wild types, the result is not readily simplified . However, the first two terms can be computed: where and are the values of prun for mutants and wild types, respectively, and ti is the probability of mutant surfing, conditioned on the event that the successful surfing attempt occurs i 1 generations after generation p. for the parameter combinations used in our simulations, mean t1 performed very well as a predictor of mean psurf for beneficial mutations (r = 96.8%), and mean t1 + t2 performed slightly better (r = 98.5%). Specifically, linear regression yielded the equations with standard errors of 0.028 for the constant and 0.056 for the coefficient of, and with standard errors of 0.019 for the constant and 0.037 for the coefficient of t1+t2 . As equations (10) and (11) show that t1 and t2 are linear functions of and, this indicates that psurf itself is very close to a linear function of these two quantities . For deleterious mutations, however, and t1+t2 drastically overestimated psurf, possibly because deleterious mutations that initially surfed later lost ground to introgressing wild types . We have developed descriptive statistical models that quantify the relationship between the probability psurf that a mutation will surf and the parameters, m and k of a frequently studied cellular automaton model (edmonds et al . We have also proposed an analytic model that performs reasonably well at predicting psurf for the case of a neutral or beneficial mutation on a one - dimensional grid . Such simplified models are desirable because a completely rigorous explanation of the dependence of surfing probability on a full range of demographic and genetic parameters currently appears unobtainable . (this is not to say that the mathematical models that have been developed, such as those in hallatschek and nelson 2008, 2009, are uninformative, but simply that they do not yield self - contained formulas for psurf as a function of system parameters in all regimes .) Moreover, a typical rigorous result for an individual - based model of invasion dynamics may simply give conditions under which the probability of persistence is either positive or zero; see, for example, the recent result on surfing in e. andjel, j. miller and e. pardoux, unpublished manuscript . In the absence of a rigorous theory, descriptive statistical models and simplified analytic models like those developed here can assess the strengths or weaknesses of heuristic explanations and may be of value in making predictions of invasiveness . Thus, such models remain crucial tools for checking and expanding upon nonquantitative verbal models of mutation dynamics during invasions . For the neutral case, our statistical models differ in several respects from those proposed in earlier work (klopfstein et al . 2005). One reason may simply be that previous authors used only grids of length 100 cells for their simulations (edmonds et al . 2004; klopfstein et al . 2005; travis et al . 2007; hallatschek and nelson 2008). This may have inflated estimates of the probability of surfing, and especially of mutant survival without surfing (travis et al . 2007 contains some discussion on this point). With the grids of length 400 used here, it is extremely rare for mutants to survive to the end of a simulation unless surfing has occurred . As a result, the bimodality of the final spatial distribution of mutants reported in edmonds et al . Some experimentation (details not shown) suggested that extension to still longer grids would have little effect on observed probability of mutant survival and psurf . We note that with the bimodality removed, it is easy to explain the midpoint rule for locating the point of origin of a mutation proposed in edmonds et al . Visual inspection of simulations in progress shows that surfing neutral mutants almost invariably occupy nearly all of the terrain from their point of origin to the rightmost boundary of the grid . It follows that the centroid of the final mutant population is halfway between the point of origin and the boundary, which is a rephrasing of the midpoint rule . In addition, as we have noted above, when beneficial mutants surf they also introgress backwards into territory previously colonized by wild types . The extent to which weak selective advantage or linkage to selected loci may bias midpoint - rule estimates therefore deserves study . More substantively, it is important to address why equations (2) and (3), which we propose as statistical descriptions of the relationship between the log - odds of surfing and the parameters, m and k, differ dramatically from the simple linear relationship between mutant success (a proxy for psurf) and the lumped parameter (1)/km proposed in klopfstein et al . (2005) (which, we note, is also consistent with the heuristics presented above). Indeed, equation (2) suggests that more appropriate lumped parameters for the one- and two - dimensional cases, respectively, might be (1)/km and (1)/k . (2005) depends on a linear regression of psurf on (1)/km . Linear regression is generally not appropriate for response variables, like psurf, that are constrained to lie between 0 and 1 (hogg and tanis 1997). (2005) it appears that much of the apparently high r attributed to this regression is due to one or a very few influential data points . Thus, we believe that equations (2) and (3) are better justified as statistical descriptions of the dependence of (and hence psurf) on, m and k. however, perhaps the most noteworthy difference between our statistical model (3) for the two - dimensional case and that proposed in klopfstein et al . (2005) is that equation (3) does not include the migration rate m. in fact, a logistic regression of on ln (m) for a 25 100 grid did show a marginally statistically significant inverse dependence [, with a standard error of 0.1077 for the coefficient of ln (m) (p = 0.090)]. (2005), which was not quantified . However, the important point is the weakness of the dependence . This is also a major difference between equation (3) and our proposed model (2) for the one - dimensional case . It is reasonable to ask what factors account for the decreased importance of m in the two - dimensional setting . One such factor may be the irregular profile of the wavefront in two dimensions (see fig . First, individuals in cells surrounded by empty or nearly empty cells (i.e. Where the wavefront has positive curvature) are expected to have more surviving offspring than individuals in cells surrounded by relatively full cells (i.e. Where the wavefront has negative curvature). Thus, the probability of surfing will be greater for mutations that happen to arise in reasons of positive curvature . Second, the irregular profile will result in competition between outcroppings of the wavefront at different latitudes in the grid . Unlike in the one - dimensional case, an outcropping of mutants at one latitude may be rivalled by an outcropping of wild types at another latitude . The mutant and wild types will eventually meet, and only one type will then fill the remaining open terrain (this has been discussed in detail in hallatschek et al . Thus, the irregular, two - dimensional wavefront introduces two additional random factors affecting the probability of surfing that do not have clear relationships to the migration rate m. contour plot of total population density in part of a 25 100 grid . Black region contains 0 individuals per cell, white region contains eight or more individuals per cell . Parameter values are = 1.8, m = 0.4, k = 10 . A point related to the relative unimportance of m in two dimensions concerns equation (6), which predicts the probability of surfing in the one - dimensional case . As noted in the analytic models section, the model performed better at smaller values of the migration rate m. this may be because the model ignores all individuals present on the grid except for those in the leading cell . In reality, individuals behind the wavefront do have some chance of filling the remaining open terrain with their progeny . To do so, however, their progeny must advance to the wavefront, and the likelihood of this occurring should decrease with m. an analogy may be drawn to solutions of fisher's equation, a classical model of travelling waves of population density (fisher 1937). The diffusion coefficient d in equation (14) is one - half the mean - squared distance traveled by an individual during one generation; so, for us d = m/2 . It is straightforward to check that near the leading edge, traveling wave solutions of equation (15) have shapes proportional to the exponential function . Thus, the number of individuals originally present one cell behind the leading cell (say) whose progeny will travel forward a given distance in a given short span of time is proportional to . This quantity increases with m. therefore, referring back to our simplified model, when an individual originally in the leading cell fails to propagate, we expect fewer candidates from cells further back to be available at the wavefront as potential propagators . As a result, ignoring all cells other than the leading cell makes equation (6) a better approximation of psurf when m is small . The simulations in the present work are necessarily limited in scope; even for the neutral case, they do not represent all possible scenarios of biological interest . In our simulations, a change in the ordering of life cycle events may change the conclusions we draw from the simulations, and it will therefore be necessary to vary this ordering in future work . Likewise, our simulations assume nearest neighbor migration in a stepping - stone model with a selection regime that is spatially and temporally invariant . If such a scenario is used as a null model for hypothesis tests regarding a population's migration history, other spatial structures and migration patterns may need to be considered as alternatives . The present work modeled only mutations arising at the leading edge of an invasion . Some authors have modeled mutations arising further back in an invasion as well (klopfstein et al . 2005; travis et al . (2007) that the probability of surfing for the model considered here drops off sharply for neutral mutations arising even a few cells behind the front; in burton and travis (2008b), it was shown that this holds true for deleterious and, to a lesser extent, beneficial mutations as well (but different boundary conditions can alter this behavior). As mutants and wild types behave identically in our neutral model, this helps explain why our simplified analytic model achieves good predictive power, even though it assumes that the probability of propagation is zero for individuals behind the front . In all the simulations discussed here, a single mutant was introduced at a fixed location once the wild - type invasion had advanced to the 10th grid column from the left . One could ask how varying mutation times and locations might affect the results . As extinction almost never occurred after the mutant was introduced, we suspect that increasing the longitude of the mutation would have little effect on psurf; however, this remains to be checked . On the other hand, changing the model to include multiple mutations, perhaps at random times, would shed light on a number of questions . Banding of the colonized territory by genotype (r. holt, personal communication), allowing for more precise tracking of a population's migration history and possibly providing the basis for a null distribution in tests for selection at other loci . Simulations with serial selected mutations could be used to study adaptive changes in quantitative traits during invasion . Among other questions, they could be used to study the distribution of effects of quantitative trait loci (qtl) in a trait subject to selection during an invasion, extending existing theory which does not take spatial population dynamics into account (mackay 2001; orr 2005). As distance from the front strongly influences the probability of surfing for neutral mutations, one might ask why we have chosen to study only mutations arising at the front . The explanation is that we have done so precisely in order to focus on what other factors may affect the probability of surfing . Analogously, one might study a group known to be at high risk of contracting a certain disease, in order to learn what additional factors might predispose an individual to contract the disease or to stay healthy . On the other hand, hallatschek and nelson have shown that as population density is (almost by definition) low at the wavefront, if mutation rates are constant over space then the modal point of origin of surfing mutations will be slightly behind the front (hallatschek and nelson 2008). Thus, studies of adaptive genetic change during invasions via the accumulation of weakly beneficial mutations need to vary their points of origin; a two - locus model which includes this variation has already been studied in burton and travis (2008a, b). As have previous studies of mutation surfing (edmonds et al . Hallatschek and nelson 2008), the present work considers only nearest - neighbor migration, in which an individual can move at most one cell length per generation . This results in diffusive movement, which can be modeled deterministically by a reaction diffusion equation; such equations are the basis for the analysis of surfing in vlad et al . However, it is well established that models assuming diffusive movement can yield inaccurate predictions when applied to many real invasions . This may be especially true when the movement rate m is relatively high for a given cell carrying capacity k. not every combination of m and k may be well described by a limiting process on a continuous spatial domain (see, for example, durrett and neuhauser 1999). A future task will be to assess, in the first instance through simulation, how patterns that have been observed for discrete mutation surfing models converge or fail to converge to limiting relationships as the scaled island model approaches a continuum . For accurate modeling, heavy - tailed dispersal kernels that model a greater frequency of long - distance dispersal events are often necessary (see, for example, kot et al . 1996 and references therein). An important area for future work will be to extend studies of mutation surfing to invasion models that incorporate heavy - tailed kernels (e.g. Laplace, or reflected exponential, kernels) and rare long - range dispersal events (this was noted in burton and travis 2008b as well). Yet, as stochastic effects inevitably will be even more important in such models, it may be extremely difficult to develop a rigorous theory that will yield useful quantitative predictions of psurf under these conditions . Simplified models that yield good agreement with simulation and, ultimately, experimental data will be especially necessary in these settings . The bulk of the present work concerns neutral mutations, but we have obtained some results for beneficial and deleterious mutations (in one - dimensional habitats) as well . Equation (4) describes the dependence of psurf on m, k and nonidentical reproduction rates mut and wt . As noted above, the direction of the dependence on m and k was the same under neutrality and selection . In addition, the psurf was much more sensitive to the value of mut than to wt, so that absolute fitness was a better predictor of surfing success than relative fitness . Consistent with this finding, equation (13), which includes as a predictor of psurf, performs only slightly better than equation (12), which does not . As it is difficult to identify consistent predictors of establishment success from empirical studies (kolar and lodge 2001; hayes and barry 2008), one might ask why analytic models that include prun (whether for mutants or wild types) as a predictor of psurf are informative . First, our results indicate that finding the determinants of surfing and of invasiveness may not be two separate hard problems but only one . Second, they lead to a novel and (in principle) empirically testable prediction about adaptive change during invasions, as follows . Our simulations indicate a strong positive association between the probability of establishment (which in this model leads to invasion) and the probability psurf of surfing, conditioned on establishment, for neutral and beneficial mutations in one dimension . (however, this association was not found for parameter regimes in which was very close to 1 .) In these simulations, the only difference between mutants and wild types was fecundity, as measured by the growth rate . For most of the parameter combinations studied here, when m and wt are held fixed and cell carrying capacity k and migration rate m are varied, higher is associated with higher psurf . This suggests that in the scenarios modeled here, conditions that favor the establishment of a population in a novel habitat might in and of themselves also favor the fixation of beneficial mutations once establishment has occurred and an invasion has begun . It has been noted previously that high intrinsic growth rates and small deme sizes at the front may promote surfing, and that genetic revolutions during invasions can occur because of surfing due to increased drift at the sparsely populated front (klopfstein et al . 2005; travis et al . 2007; excoffier and ray 2008; hallatschek and nelson 2008, 2009), but the finding that factors other than and population density can promote surfing in some invasions is novel, to our knowledge . As k and m do not depend solely on organismal traits (whether genetic or nongenetic) but also on features of a given habitat, our results suggest that even abiotic factors that promote establishment might also promote the survival and spread of beneficial mutations (as well as neutral ones), conditioned on establishment . This finding is complementary to studies that have identified propagule size and number of introductions as the factors with the strongest currently known association with establishment success (kolar and lodge 2001; hayes and barry 2008). Further simulation and empirical studies seem warranted to assess the generality of this effect and its importance in explaining adaptive genetic change during invasions.
Lipomas are the most common benign mesenchymal tumors of the gastrointestinal tract, with the colon being the most prevalent site . Tumors> 2 cm in diameter may occasionally cause nonspecific symptoms, including change in bowel habits, abdominal pain, or rectal bleeding, but with resection the prognosis is excellent . Herein, we describe the case of an elderly male who presented with painless hematochezia . Both colonoscopy and computed tomography of the abdomen and pelvis confirmed the presence of a mass near the ileocecal valve . Because of continuing bleeding, the patient required laparoscopic - assisted right hemicolectomy to resect the mass . Previous cases of ileocecal valve lipomas have been reported in the english literature, with the majority presenting as intussusception or volvulus . We present a rare case of an ulcerated ileocecal valve lipoma presenting as lower gastrointestinal bleeding that was treated successfully with laparoscopic resection . Distribution of alimentary lipomas demonstrates a predilection for the colon, but they may originate anywhere in the gastrointestinal tract, from the hypopharynx to the rectum . Often asymptomatic and detected incidentally at the time of colonoscopy or surgery, lipomas> 2 cm in diameter may occasionally cause nonspecific symptoms, including change in bowel habits, abdominal pain, or rectal bleeding . Diagnosis of gastrointestinal lipomas may involve barium enema, colonoscopy, or computed tomography (ct). Diagnosis is notoriously difficult given that malignant disease cannot be excluded definitively through imaging or biopsy alone . A wide range of operative and nonoperative techniques has been utilized for resection . Few cases of lipoma have been reported to date with origin at the ileocecal valve, the majority of which have presented as intussusception or volvulus . We describe a rare case of an ulcerated ileocecal valve lipoma associated with lower gastrointestinal bleeding that was significant enough to require urgent laparoscopic resection . A 77-year - old male with a history of diabetes mellitus, hypertension, coronary artery disease, aortic valve replacement, and coronary artery bypass grafting, who was therapeutic on warfarin, presented to his gastroenterologist with 2 days of painless hematochezia . Outpatient colonoscopy performed at that time revealed a 6-cm cecal mass with mucosal necrosis at the ileocecal valve, as well as a small ulcer at the hepatic flexure . Given the colonoscopic findings, the patient was transported urgently to our emergency department . Upon presentation, the patient was in no acute distress with a core temperature of 36.4c, heart rate of 73 beats/ min, blood pressure of 135/91 mm hg, and respiratory rate of 18 breaths / minute . His abdomen was soft, nontender, and nondistended with no evidence of peritoneal irritation . Computed tomography of the abdomen and pelvis with oral and intravenous contrast revealed a 1.9-cm x 1.5-cm lesion within the cecum (figure 1). The patient was admitted for hemodynamic monitoring, transfusion, and surgical resection of the ileocecal mass . Ct image of mass preoperatively, demonstrating a 1.9-cm 1.5-cm lesion within the cecum (arrow), measuring -80 hounsfield units, consistent with adipose tissue . The patient received vitamin k and 2 units of fresh frozen plasma for reversal of the effect of warfarin . Nonetheless, hematochezia continued with a steady decrease in hematocrit from 43% to 39% over a 24-hour period . On hospital day 2, he underwent an uncomplicated laparoscopic - assisted right hemicolectomy and ileo - transverse colostomy . Pathology revealed a 3.5-cm x 2.0-cm x 1.5-cm mass at the ileocecal valve (figure 2). The yellow, homogeneous cut surfaces and the microscopic evaluation (figure 3) were both consistent with lipoma of the ileocecal valve . The patient regained bowel function by postoperative day 4 and was discharged the following day . Intraoperative photograph with cecum opened to reveal a 3.5-cm 2.0-cm 1.5-cm tumor (bottom arrow) and ileocecal valve (top arrow). Histopathology of lipoma: hematoxylin and eosin stain (20 magnification) reveals a circumscribed collection of mature adipocytes . Gastrointestinal lipomas are benign lesions arising from the adipocytes within the intestinal submucosa with the first case reported by bauer in 1757 . With a reported incidence of 0.2% to 4.4%, lipomas are the third most common benign colonic neoplasm following hyperplastic and adenomatous polyps . Lipomas are found most commonly in the colon, with the highest frequency found in the ascending colon and cecum followed by the transverse colon, descending colon, sigmoid, and least often the rectum . Despite the propensity for colonic distribution, lipomas can occur anywhere along the alimentary tract, including the hypopharynx, stomach, small bowel, and esophagus . When confined to the colon, 90% of these lesions are localized to the submucosa; however, a few reports have suggested an origin in the subserosal plane . These tumors are more prevalent in women and are typically discovered during the fifth or sixth decade of life . They vary in size and can be sessile or pedunculated . Cases of multiple lesions have been reported . Lipomas are generally asymptomatic and are identified most commonly as incidental findings during colonoscopy, surgery, or autopsy . In the minority of patients who do present with symptoms, the lesions tend to be> 2 cm in diameter, as in the present case . Common symptoms include constipation, diarrhea, colicky abdominal pain, or lower gastrointestinal bleeding . Abdominal pain may be associated with intermittent intussusception, whereas gastrointestinal bleeding is secondary to ulceration of the overlying mucosa . In rare cases, patients can present with dramatic clinical symptomatology that requires urgent operative intervention, usually for intussusception or acute hemorrhage . Our patient presented with hematochezia likely secondary to the combination of the size, mucosal necrosis at the surface of the lipoma, and the patient's anticoagulation regimen . A variety of imaging modalities are available to assist in the preoperative diagnosis of gastrointestinal lipoma . Barium enema shows colonic lipomas as ovoid, well - delineated, and smooth radiolucent masses . A squeeze sign can also be noted, which indicates a change in size and shape of the lesion due to peristalsis . Computed tomography scanning is a second modality that provides a more definitive diagnosis in uncomplicated cases, where lipomas appear as sharply demarcated ovoid lesions with absorption densities of -40 to -120 hounsfield units . In the present case, lastly, endoscopy is a third diagnostic tool with 2 typical findings: tenting is described when the mucosa overlying the lipoma is easily retracted away from the mass with biopsy forceps, and a cushion sign is present when the forceps produces a soft, cushioning indentation when applied to the lipoma . Due to the submucosal location of these lipomas, rarely, colonoscopy can reveal ulcerations and a lack of the cushion sign, which may lead to the impression of malignant disease as in the case of our patient . Katsinelos et al described 11 lesions that demonstrated malignant features on colonoscopy but were proved ultimately to be benign lipomas on histopathology examination . Preoperative diagnosis of gastrointestinal lipomas can be difficult when it presents as signs and symptoms suggesting malignant disease that cannot be excluded definitively through imaging or biopsy alone . The greatest clinical importance of intestinal lipoma is its potential to be confused with malignant colonic neoplasm . Immediate surgical intervention is mandatory in cases of obstruction, intussusception, perforation, or massive hemorrhage with the last sign being seen in our patient . Several operative and nonoperative techniques have been described, including laparotomy, mini - laparotomy, and laparoscopy to perform enucleation, colostomy, excision, or segmental colonic resection of lipomas . Among the nonoperative techniques, endoscopic removal of symptomatic lipomas is controversial due to the inefficient conduction of electric current through adipose tissue . This inefficiency results in an unacceptably high rate of complications, including perforation or hemorrhage . Previous case reports have demonstrated that ileocecal valve lipomas present most commonly as intussusception or volvulus . Only one of these earlier cases was managed by laparoscopic resection . To our knowledge, laparoscopic resection has not been utilized in the setting of acute hemorrhage secondary to an ileocecal valve lipoma . However, given that lipomas disbursed throughout other regions of the large intestine have been resected successfully through laparoscopy, laparoscopic resection may now be considered an excellent, minimally invasive option for the treatment of ileocecal valve lipomas presenting as intussusception, volvulus, or hemorrhage . We present a case of ileocecal valve lipoma in a patient who presented with acute gastrointestinal hemorrhage . After an extensive literature review, it was found that the majority of intestinal lipomas present as colonic lipomas with either intussusceptive or obstructive symptoms . We believe this is a very rare case of an ileocecal valve lipoma presenting as ulcerations and necrosis leading to acute hemorrhage and urgent resection.
Suprapubic cystostomies are performed for a variety of indications that require either temporary or permanent placement of suprapubic catheters . The placement of percutaneous suprapubic catheters using a punch trocar technique involves some risks and should not be considered an innocuous procedure . Among the wide variety of complications, the most serious complication is bowel injury . Bowel injuries include perforation of the ileum [2 - 6] and colon as well as small bowel obstruction and volvulus [3,7 - 9]. Because the percutaneous tract is blindly made at 3 to 4 cm above the symphysis pubis in the midline, the risk of bowel injury is constantly present . Therefore, before a percutaneous cystostomy is performed, especially if there has been prior abdominal or pelvic surgery, or if the bladder is not full, it is strongly advised to consider using ultrasound for the detection of interposed bowel along the percutaneous tract, because the bowel may be in close proximity to the tract . However, that method does not totally guarantee the nonexistence of bowel crossing the tract, because it may be difficult for the ultrasound to detect a collapsed bowel . Despite this clinical setting, there has been little study of the risk factors for bowel injury associated with percutaneous cystostomy . This may be largely due to the practical limitations in demonstrating bowel injury before and after suprapubic cystostomy . In this study, we investigated the risk factors for possible bowel interposition between the bladder and the skin at the routine puncture site of suprapubic cystostomy by computed tomography (ct), assuming that a hypothetical suprapubic cystostomy was performed . We retrospectively reviewed 795 consecutive adult abdominopelvic ct scans performed for various reasons in our hospital from september to october 2009 . Among them, we selected 226 patients whose bladders were distended more than 6 cm above the upper margin of the symphysis pubis . Their medical charts at the time of the ct scan were analyzed retrospectively . Through the ct scan images, we determined whether the bowel was interposed between the urinary bladder and the skin at a point along the midline of the abdomen, 3 cm above the upper margin of the symphysis pubis (fig . We investigated whether the age, gender, height, history of radical pelvic surgery, or the distance between the upper margin of the symphysis pubis and the umbilicus influenced the possibility of bowel interposition between the bladder and the skin at the suprapubic puncture site . Statistical analyses were performed by applying the chi - square test and multiple logistic regression using commercially available software spss ver . 14.0 (spss inc ., chicago, il, usa). A p - value of <0.05 was considered statistically significant . The patients' mean age was 63 years (range, 26 - 84 years). Among the 226 patients, 22 (9.7%) had undergone radical pelvic surgery previously . The mean distance between the upper margin of the symphysis pubis and the umbilicus was 14.4 cm (range, 7.2 - 21.0 cm) (table 1). The univariate analysis showed that sex (female, p=0.028), obesity (body mass index [bmi]25 kg / m, p=0.006), a positive history of radical pelvic surgery (p<0.001), and a short distance (11 cm, p=0.016) between the upper margin of the symphysis pubis and the umbilicus were significantly correlated with bowel interposition at the routine suprapubic puncture site (table 2). Among the 22 patients who had a radical pelvic operation, 20 had lower anterior resection, and the other 2 had segmental resection of the rectum due to rectal or colon cancer . In the multivariate analysis, obesity (p=0.019), a positive history of pelvic surgery (p=0.001), and a short distance (p=0.016) between the upper margin of the symphysis pubis and the umbilicus were independent predictors for the bowel passing through the percutaneous tract (table 3). In our multivariate analysis, obesity, a history of pelvic surgery, and a short distance between the symphysis pubis and the umbilicus were independent predictors of bowel interposition through the percutaneous suprapubic cystostomy tract . Prior pelvic surgery is a well known risk factor for bowel injury after percutaneous cystostomy . After a pelvic operation, the bowel can move downward through the opened space of retzius, and associated postoperative adhesions may develop between the anterior abdominal wall and peritoneum, both of which may increase the probability of bowel injury during a suprapubic cystostomy procedure . In our study, 9 of the 22 patients (40.9%) who had radical pelvic surgery had an interposed bowel at the routine suprapubic cystostomy tract, whereas 17 of the 204 (8.3%) without a history of pelvic surgery had bowel interposition . All of the prior pelvic surgeries were opened lower anterior resections because of rectal or colon cancer . Obesity has also been considered as a risk factor for bowel injury when performing a suprapubic cystostomy because it is difficult to localize the bladder in such patients . Our study presented the novel hypothesis that obesity itself is an independent predictor for bowel interposition through the cystostomy tract . We do not know the exact reason for the above finding, but assumed that because obese patients have relatively larger bowel contents as well as omental fat, the bowel tended to move downward in the limited intra - abdominal space . Similarly, the correlation of bowel interposition with a short distance between the symphysis pubis and the umbilicus can be understood in the same manner . As we mentioned above, 8.3% of the patients without a history of pelvic surgery had interposition of the bowel through the routine cystostomy tract . This was much higher than the general consensus, because the placement of a suprapubic tube for urinary diversion is generally an uneventful procedure . For the safe placement of a suprapubic catheter, the most important point is the full distension of the urinary bladder before the procedure . In this study, we considered bladder distension more than 6 cm above the upper margin of the symphysis pubis as a cutoff point on the basis of data from voiding cystourethrograms performed in 20 adult patients with neuropathic bladder . In their films, the average distance between the symphysis pubis and the upper margin of the cystogram at full bladder distension was 6.2 cm . We therefore defined 6 cm as the assumptive point of full bladder distension . Because a fully distended bladder in a normal patient without neuropathic bladder can rise more than 6 cm above the symphysis pubis, it seemed likely that our results overdetected the bowel interposition through the cystostomy tract . However, we should take into consideration that, clinically, the complications of suprapubic cystostomy are commonly seen in small capacity bladder with neuropathic deficits . In the practical setting, although a small capacity bladder itself would be the major risk factor for complications in performing suprapubic cystostomy, the retrospective nature of our study could not reveal that relationship . The high exclusion rate (71.6%) was another major pitfall of this study, because patients who underwent abdominopelvic ct scan were not instructed to endure the urge to urinate . To our knowledge, this is the first study that attempted to reveal the risk factors of possible bowel injury related to suprapubic cystostomy, even though the study was performed by an indirect approach . This study demonstrated the risk factors for the bowel being interposed at the routine suprapubic puncture site as detected by ct scan . When performing a suprapubic cystostomy, extreme caution is needed to avoid possible bowel injury in patients who are obese, who have had previous radical pelvic operations, and who have a short distance (11 cm) between the upper margin of the symphysis pubis and the umbilicus.
The study participants from the usa are members of a colorectal cancer prognosis study (colocare), with sites at the fred hutchinson cancer research center, seattle (wa, usa), h. lee moffitt cancer center and research institute, tampa (fl, usa), and the national center for tumor diseases, german cancer research center, heidelberg (germany). The objective of our consortium is to identify the factors that determine short - term survival in a prospective cohort of colorectal cancer patients . These include inherited genetic and tumour characteristics that affect early detection, treatment response and prognosis as well as health behaviours that patients adopt that may have health outcomes . German samples included in this pilot study were obtained from participants of the darmkrebs chancen der verhtung durch screening (dachs) study at the german cancer research center in heidelberg (germany). Samples included from tampa were collected as part of moffitt's total cancer care program . Eligible for the study were colorectal cancer patients, at least 18 years of age, with a first diagnosis of invasive colon or rectal cancer (stages i iv) receiving care at any of the colocare consortium / dachs sites . This pilot study involved 149 colorectal cancer cases and ninety - one age- and sex - matched controls (heidelberg, forty cases and forty controls; seattle, ninety - one cases and thirty - three frequency - matched controls; tampa, eighteen cases and eighteen controls), recruited between the years 2006 and 2010 and whose biospecimens were obtained within the same time frame with identical blood - draw protocols at each of the sites . Blood samples were obtained at the time of recruitment into the study . For the cases, the samples were pre - treatment samples obtained at the time of surgery . In the dachs study, control subjects were community - based and randomly selected from population registries . They were frequency - matched to cases on age, sex and county of residence . We excluded controls with a history of colorectal cancer; otherwise eligibility criteria were the same as for the cases . Controls were contacted through mail and follow - up calls, and interviews were conducted at their homes . The participation rate among eligible control participants was a little higher than 50% . The controls in moffitt, tampa were frequency matched to the cases on age (5-year intervals), sex and race (white non - hispanic). Controls in seattle were drawn from participants in a screening and surveillance colonoscopy programme who had no evidence of pathology within the colon and were matched to the cases for age, sex and race . This study was conducted according to the guidelines laid down by the declaration of helsinki and all procedures involving human subjects were approved by the institutional review boards of the german cancer research center (dkfz), the fred hutchinson cancer research center, seattle (wa, usa) and h. lee moffitt cancer center and research institute, tampa (fl, usa). Plasma 25(oh)d3 was measured by reverse - phase hplc in the biochemistry and biomarkers laboratory of the division of preventive oncology, german cancer research center, dkfz (dr owen). The intra - assay and inter - assay cv of 25(oh)d3 at a concentration of 50 ng / ml were 23 and 31%, respectively . Snb 57co / i radioassay kit (mp biomedicals) in the biomarker laboratory (dr song) at the fred hutchinson cancer research center with intra - assay cv of 31 and 35% and inter - assay cv of 70 and 51% for folate and vitamin b12, respectively . To account for skewed distributions, wilcoxon or kruskal wallis tests were used to test for the differences in biomarker concentration by age group (1859, 6069 and 7080 years), case status, tumour stage (i iv), study site (heidelberg, seattle and tampa), and season of blood draw (spring: march may; summer: june august; autumn: september november; winter: december february). Because the samples were unequally distributed across seasons at the three study sites, we computed season - corrected levels for the descriptive analyses of 25(oh)d3 in order to give equal weight to all four seasons . Linear regression analysis (using both forward stepwise and enter methods) with log - transformed outcome parameters was used for multivariate analyses . All statistical analyses were conducted using sas (v9.1.2, sas institute inc . ). Statistical significance was defined as p <005 and all statistical tests were two - sided . Cases and controls were, on average, aged 600 (sd 133) and 609 (sd 131) years, respectively . The majority of tumours were of stages iii (396%) and iv (262%). Among the cases, the mean 25(oh)d3, folate and vitamin b12 concentrations were 256 (sd 135) ng / ml, 216 (sd 140) ng / ml, and 638 (sd 597) pg / ml, respectively, whereas among the controls, they were 259 (sd 140) ng / ml, 180 (sd 137) ng / ml, and 515 (sd 241) pg / ml (table 1). Table 1.baseline characteristics of colorectal cancer cases and controlscases (n 149)controls (n 91)characteristic n% n% study siteheidelberg4026840440seattle9161133363tampa1812118198age (years)mean6061 sd 1313185964433640606947323235709038262325sexmale80544853female69464347racewhite, non - hispanic137928492non - white or other12878tumour stagei (i, ia, ib and ic)20134ii (ii, iia, iib and iic)30201iii (iii, iiia, iiib and iiic)59396iv39262unknown107season of blood drawspring (march may)2718123253summer (june august)3221525275autumn (september november)4429523253winter (december february)4630920220nutritional biomarker25(oh)d3 (ng / ml)mean256259 sd 135140range4680040830folate (ng / ml)mean216180 sd 140137range0370314625vitamin b12 (pg / ml)mean638515 sd 597241range96375094135225(oh)d3, 25-hydroxyvitamin d3 . Baseline characteristics of colorectal cancer cases and controls 25(oh)d3, 25-hydroxyvitamin d3 . In multivariate modelling, significant predictors for the biomarkers included season for 25(oh)d3, study site for 25(oh)d3, folate, vitamin b12, and tumour stage for vitamin b12 (table 2). Probably because of the limited sample size of this pilot study, case status was not statistically significant as a predictor; hence, cases and controls were combined in subsequent analyses . Also, the results from models excluding case - status were identical to those including case - status in the model . The association from controls only was similar, yet did not reach statistical significance . Plasma 25(oh)d3 concentrations were, on average, higher in heidelberg (317 ng / ml; range 110830 ng / ml) than in seattle (233 ng / ml; range 40800 ng / ml) and tampa (211 ng / ml; range 46516 ng / ml) (table 3). Hypovitaminosis d (plasma 25(oh)d3 <20 ng / ml) was more prevalent in tampa (54%) and seattle (50%) than in heidelberg (23%) (data not shown). On the other hand, plasma folate concentrations were, on average, lower in heidelberg (111 ng / ml; range 03482 ng / ml) than in seattle (253 ng / ml; range 35703 ng / ml) and tampa (238 ng / ml; range 68697 ng / ml). No participants from seattle and tampa had folate deficiency (folate <3 ng / ml), while 10% of those from heidelberg had folate deficiency . Plasma vitamin b12 concentrations followed the same pattern as folate with lower average values recorded in heidelberg (395 pg / ml; range 961641 pg / ml) than in seattle (740 pg / ml; range 1833750 pg / ml) and tampa (522 pg / ml; range 941085 pg / ml). Table 2.predictors of plasma 25-hydroxyvitamin d3 (25(oh)d3), vitamin b12 and folate among colorectal cancer cases and controlsbiomarkerpredictordirectionsignificance25(oh)d3heidelberg+summer season+autumn season+vitamin b12heidelbergstage iv+folateheidelbergage 6069 years+ * p <010, * * p <005.cases and controls were combined because case status was not statistically significant . Table 3.nutritional biomarker concentrations among colorectal cancer cases and controls in the three study locations(mean values and ranges)heidelbergseattletampameanrangemeanrangemeanrange25(oh)d3 (ng / ml)317110, 83023340, 80021146, 516folate (ng / ml)11103, 48225335, 70323868, 697vitamin b12 (pg / ml)39596, 1641740183, 375052294, 108525(oh)d3, 25-hydroxyvitamin d3 . Predictors of plasma 25-hydroxyvitamin d3 (25(oh)d3), vitamin b12 and folate among colorectal cancer cases and controls * p <010, * * p <005 . Cases and controls were combined because case status was not statistically significant . Nutritional biomarker concentrations among colorectal cancer cases and controls in the three study locations (mean values and ranges) 25(oh)d3, 25-hydroxyvitamin d3 . Seasonal differences in plasma 25(oh)d3 concentrations were evident in heidelberg, where the highest plasma 25(oh)d3 concentrations were observed in summer (370 ng / ml) and the lowest in spring (213 ng / ml), but no such seasonal variation was noted in seattle and tampa (fig . 1. (a) plasma vitamin d (25-hydroxyvitamin d; 25(oh)d) concentration by study site . The horizontal bars represent medians and the circles represent potential outliers in the data . (d) seasonal plasma 25(oh)d by study site:, spring;, summer;, autumn;, winter . Values are means . (a) plasma vitamin d (25-hydroxyvitamin d; 25(oh)d) concentration by study site . The horizontal bars represent medians and the circles represent potential outliers in the data . (d) seasonal plasma 25(oh)d by study site:, spring;, summer;, autumn;, winter . To the best of our knowledge, this is the first study to compare a range of nutritional biomarkers (25(oh)d3, folate and vitamin b12) among colorectal cancer patients and controls in different geographical regions . Our international pilot study revealed differences in biomarker concentrations between the us sites and germany . Because dietary sources of vitamin d are very limited, cutaneous synthesis of vitamin d (within the uvb wavelength of 290315 nm) is a very important source of plasma 25(oh)d3 . The uv radiation required for the synthesis of vitamin d in the skin is more abundant and more functional during summer than during winter . Hence, among people who do not use adequate vitamin d supplements or consume vitamin d - fortified foods, season is one of the most important determinants of plasma 25(oh)d3 concentrations . Thus, the seasonal variations in plasma 25(oh)d3 observed in heidelberg are in line with expectations . Somewhat surprising is the lack of seasonal variation and the low plasma 25(oh)d3 concentrations observed in seattle and tampa . Of the three sites, tampa has the highest daytime temperatures during summer but the participants from tampa had the lowest plasma 25(oh)d3 concentrations during summer . The differences in plasma 25(oh)d3 concentration during summer among participants in our study could reflect the extent of outdoor activities and possible sun - avoidance behaviours (including sunscreen use) in the three cities . In heidelberg, people are more likely to commute with bicycles or walk than use cars, whereas people are more likely to practise sun - avoidance in florida . Few studies have examined the impact of season on 25(oh)d3 concentrations in lower altitude regions in the usa . Although a previous study in south florida reported a seasonal variation in serum 25(oh)d3 concentrations, the magnitude (14%) is much smaller than what is observed (38%) among residents of northern latitudes in the usa . Compared with the previous study from south florida, the winter 25(oh)d3 concentrations among participants from tampa in our study are identical (233 v. 231 ng / ml, respectively) but the summer concentrations were lower (177 v. 268 ng / ml). Thus, the low summer 25(oh)d3 concentrations in tampa could be a chance finding due to the low number of participants from tampa in our study . Studies have revealed that current food fortification practices in the usa may not be adequate to prevent vitamin d deficiency among vulnerable people during winter . Likewise, it has been shown that although supplement use increases serum 25(oh)d3 concentrations, it does not make up for insufficient sun exposure, and current supplement recommendations are not adequate to prevent vitamin d deficiency . As vitamin d supplementation is discussed in the context of improving survival in colorectal cancer patients, our study shows that high plasma vitamin d concentrations can be achieved in populations with substantial outdoor activity during summer (such as heidelberg). Nevertheless, vitamin d fortification in the usa did not prevent low levels of serum 25(oh)d3 in tampa or seattle, cities that practise sun - avoidance or reduced sun exposure . The lower plasma folate concentrations among participants from heidelberg compared with those from the usa are not unexpected considering the fact that mandatory fortification of staple foods with folic acid commenced in the usa in 1998 while fortification is not practised in germany . The mandatory fortification programme has resulted in a substantial increase in plasma folate concentrations in the usa, where the prevalence estimates for low serum folate concentrations (<3 ng / ml) declined from 206% in the period 19881994 to 06% in 2004 . However, we also observed large intra - site differences in plasma folate and vitamin b12 concentrations at each study site . This may be due mostly to the use of folic acid - containing supplements . It has been shown that supplement and multivitamin use is higher among cancer patients compared with the general population . Between 64 and 81% of cancer patients use any vitamin or mineral supplement, compared with 50% in the general population . In a large study in washington state, usa, colorectal cancer patients were 33% more likely to use folic acid supplements compared with cancer - free controls and such a supplement use is associated with higher plasma folate and vitamin b12 concentrations . Therefore, the use of folic acid - containing supplements is an important determinant of plasma folate concentrations in colorectal cancer patients . This may be of potential concern considering the fact that very high plasma folate concentrations that can be achieved with supplement use may accelerate the growth of already existing colorectal cancers . In a randomised controlled trial, folic acid given at a dosage of 1 mg / d was associated with a 17-fold increased risk of advanced colorectal cancers, thus excluding its utility and safety as a secondary or tertiary prevention agent for colorectal cancer . However, this is an area of ongoing research and further studies are needed to elucidate the relationship between folate and survival in patients with colorectal cancer . A particular strength of our study is the comparison of biomarkers across international study sites with identical laboratories for assays and consistent blood - draw protocols . However, our sample size as well as variable set was limited and we had no information on supplement use; hence we could not investigate the association of supplement use with nutritional biomarker concentrations . In conclusion, we observed substantial differences in nutritional biomarker concentrations across international study sites, probably reflecting dissimilarities in supplement use, food fortification, and sun - seeking behaviour, among other health practices . Intra - site differences at each study location were larger than between - location variability, suggesting that individual health behaviours play a significant role . As we aim to understand predictors of colorectal cancer prognosis, there is much to learn from international cohorts, which offer a broad range of exposure and health behaviours that cannot be obtained at a single site.
Corneal collagen cross - linking (cxl) with riboflavin and ultraviolet - a (uv - a) is currently the only treatment that can halt or delay the progression of keratoconus . In this technique, the combined action of riboflavin and uv - a rays is utilized to increase the corneal strength and integrity by polymerization of the stromal fibers.12 with the widespread adoption of this technique, reports of side - effects and complications are increasing.34567 in this case report, we present two patients who developed clinically diagnosed herpetic keratitis in the early postoperative period after cxl . There was no previous history of intraocular or corneal surgery, herpetic keratitis, autoimmune disease or systemic connective tissue disease . The uncorrected visual acuity (ucva) was 20/100 in the right eye and 20/70 in the left eye . The best spectacle - corrected visual acuity (bscva) was 20/40 in the right eye and 20/25 in the left eye . The central pachymetry was 436 m oculus dexter (od) and 457 m oculus sinister (os). Topography after 6 months showed the progression of keratoconus (defined as either an increase in the manifest cylinder of 1 d or greater or an increase in the steepest keratometry by 1 d or greater). The recommended treatment was cxl with riboflavin and uv - a to stabilize the cornea . The patient underwent a thorough discussion of the risks and benefits of cxl with the surgeon and signed a written informed consent in accordance with institutional guidelines based on the declaration of helsinki . A 9.0 mm diameter of the corneal epithelium was mechanically removed using a surgical blade, and riboflavin 0.1% solution was instilled repeatedly for approximately 30 min . Penetration of the cornea and presence of riboflavin in the anterior chamber (riboflavin shielding) was confirmed by slitlamp examination . Uv - a irradiation was performed using an optical system (uv - x illumination system version 1000; avedro inc ., waltham, ma, usa) with a light source consisting of an array of uv diodes (365 nm) with a potentiometer in series to allow regulation of voltage . Irradiance of the cornea with 3 mw / cm was performed for 30 min . During treatment, the riboflavin solution was applied every 2 min and drops of the physiological saline solution were instilled in between to moisten the cornea . After treatment, postoperative medication included predinsolone acetate 1% (pred forte; allergan inc ., irvine, ca, usa) four times daily tapered over 4 weeks, ofloxacin 0.3% (optiflox; jamjoom pharma, oman) four times daily for 2 weeks, and cyclopentolate 1% tid for 1-week . The patients were also advised to instill preservative free artificial tears (tears natural free; alcon inc ., hnenberg, switzerland). On the 7 postoperative day, the patient presented with a geographic epithelial defect with dendritic edges without iritis . Topical corticosteroid was discontinued, and the patient was started on ganciclovir (virgan gel, 1.5 mg / g; thea pharma, wetteren, belgium) five times daily for 2 weeks then three times daily for 2 weeks . After 2 days of initiation of topical antiviral therapy, the geographic ulcer dramatically improved and re - epithelialized [figure 1]. Four months later, a mild central corneal opacity remained but the ucva recovered to preoperative levels without any recurrence of keratitis [figure 2]. Corneal dendrite stained with fluorescein after 2 days of treatment (photo was not taken at the beginning of the treatment) mild corneal haze after 4 months of treatment a 21-year - old male presented with progressive bilateral keratoconus . The patient was healthy without any history of systemic diseases or previous ocular surgery . The patient underwent a thorough discussion of the risks and benefits of cxl with the surgeon and signed a written informed consent in accordance with institutional guidelines based on the declaration of helsinki . Corneal crosslinking of the right eye was performed under sterile conditions using the same technique and the same postoperative medication that was described above . On the 6 day, slow re - epithelialization was observed . On the ninth postoperative day, topical corticosteroids were discontinued, and the patient was started on ganciclovir virgan ((ganciclovir 0.15%) eye gel, thea pharmaceuticals ltd, newcastle, uk) eye gel five times daily . Dendritic epithelial keratitis (on the 9 day postcorneal collagen cross - linking) mild corneal haze after 4 months of treatment there was no previous history of intraocular or corneal surgery, herpetic keratitis, autoimmune disease or systemic connective tissue disease . The uncorrected visual acuity (ucva) was 20/100 in the right eye and 20/70 in the left eye . The best spectacle - corrected visual acuity (bscva) was 20/40 in the right eye and 20/25 in the left eye . The central pachymetry was 436 m oculus dexter (od) and 457 m oculus sinister (os). Topography after 6 months showed the progression of keratoconus (defined as either an increase in the manifest cylinder of 1 d or greater or an increase in the steepest keratometry by 1 d or greater). The recommended treatment was cxl with riboflavin and uv - a to stabilize the cornea . The patient underwent a thorough discussion of the risks and benefits of cxl with the surgeon and signed a written informed consent in accordance with institutional guidelines based on the declaration of helsinki . A 9.0 mm diameter of the corneal epithelium was mechanically removed using a surgical blade, and riboflavin 0.1% solution was instilled repeatedly for approximately 30 min . Penetration of the cornea and presence of riboflavin in the anterior chamber (riboflavin shielding) was confirmed by slitlamp examination . Uv - a irradiation was performed using an optical system (uv - x illumination system version 1000; avedro inc ., waltham, ma, usa) with a light source consisting of an array of uv diodes (365 nm) with a potentiometer in series to allow regulation of voltage . Irradiance of the cornea with 3 mw / cm was performed for 30 min . During treatment, the riboflavin solution was applied every 2 min and drops of the physiological saline solution were instilled in between to moisten the cornea . After treatment, postoperative medication included predinsolone acetate 1% (pred forte; allergan inc ., irvine, ca, usa) four times daily tapered over 4 weeks, ofloxacin 0.3% (optiflox; jamjoom pharma, oman) four times daily for 2 weeks, and cyclopentolate 1% tid for 1-week . The patients were also advised to instill preservative free artificial tears (tears natural free; alcon inc ., hnenberg, switzerland). On the 7 postoperative day, the patient presented with a geographic epithelial defect with dendritic edges without iritis . Topical corticosteroid was discontinued, and the patient was started on ganciclovir (virgan gel, 1.5 mg / g; thea pharma, wetteren, belgium) five times daily for 2 weeks then three times daily for 2 weeks . After 2 days of initiation of topical antiviral therapy, the geographic ulcer dramatically improved and re - epithelialized [figure 1]. Four months later, a mild central corneal opacity remained but the ucva recovered to preoperative levels without any recurrence of keratitis [figure 2]. Corneal dendrite stained with fluorescein after 2 days of treatment (photo was not taken at the beginning of the treatment) mild corneal haze after 4 months of treatment a 21-year - old male presented with progressive bilateral keratoconus . The patient was healthy without any history of systemic diseases or previous ocular surgery . The patient underwent a thorough discussion of the risks and benefits of cxl with the surgeon and signed a written informed consent in accordance with institutional guidelines based on the declaration of helsinki . Corneal crosslinking of the right eye was performed under sterile conditions using the same technique and the same postoperative medication that was described above . On the 6 day, slow re - epithelialization was observed . On the ninth postoperative day, topical corticosteroids were discontinued, and the patient was started on ganciclovir virgan ((ganciclovir 0.15%) eye gel, thea pharmaceuticals ltd, newcastle, uk) eye gel five times daily . Dendritic epithelial keratitis (on the 9 day postcorneal collagen cross - linking) mild corneal haze after 4 months of treatment corneal collagen cross - linking with riboflavin and uv - a is becoming the accepted method for the treatment of progressive keratoconus.3 it has been shown to increase effectively the biomechanical strength of the cornea and halt the progression of keratoconus . Reactivation of herpes simplex virus (hsv) has been reported after emotional stress, trauma, fever, and laser surgery.4 these established clinical triggers are thought to be mediated by the adrenergic and sensory nervous systems . Exposure to uv light can also induce oral and genital herpes in humans and ocular herpes in animal models.568 the herpetic eye study group9 studied many triggers including, psychological stress, systemic infection, sunlight exposure, menstrual cycle, and contact lens wear . None of these factors were significantly associated with recurrence of herpetic keratitis . In this case report, we describe two patients who developed typical dendritic herpetic epithelial keratitis after cxl treatment . In our cases, the presumed clinical diagnosis of herpetic keratitis was supported by the clinical appearance of the lesions, delayed epithelial healing, decreased corneal sensation and the rapid response to antiviral treatment . In our cases, we typically follow the patient carefully until the epithelium is intact; delayed healing raises the suspicion of secondary infective keratitis . Therefore, we propose that it is important to closely monitor the patient until there is complete reepithelialization . Also, in cases of delayed healing secondary infective keratitis should be ruled out . Uv - a light may be a potent stimulus to trigger reactivation of latent hsv infections, even in patients with no history of clinical ocular herpes infection . Significant corneal epithelial / stromal trauma or actual damage of the corneal nerves could be the mechanism for hsv reactivation . The use of topical corticosteroids with an epithelial defect is an additional risk factor for infectious keratitis of any etiology . In our cases, both patients did not have a history of previous herpetic eye disease or cold sores, which concur with kyminonis's et al yuksel et al.10 also reported a no previous history of herpetic keratitis or cold sores . Primary hsv keratitis frequently manifests as a nonspecific upper respiratory tract infection and is recognized as hsv <5% of the time . Latent infection of the trigeminal ganglion may occur in the absence of recognized primary infection, with reactivation occurring later as recurrent hsv infection in any branch of the trigeminal nerve . Secondary hsv infection can affect almost any ocular tissue including: the eyelid, conjunctiva, cornea, iris, trabecular meshwork, and retina . Secondary hsv can occur as a self - limited disease, and patients may not seek medical advice in some episodes . Our patients presented later than in previous reports.710 there were no recurrences in both patients for 4 months, which was not been documented in previous case reports.710 their diagnosis was confirmed with polymerase chain reaction tear analysis for hsv . Despite the rapid closure of the epithelium within 2 days and initiation of topical steroids, they also noted a mild central corneal scar at 2 months which were similar to our patients . As cxl is becoming increasingly popular for the treatment of progressive keratoconus, new side - effects and complications are emerging . Herpetic keratitis may be triggered by cxl, even in cases with no history of the disease . Close follow - up of patients after cxl appears to be important, as early diagnosis and proper treatment can facilitate the successful management of herpetic epithelial keratitis and prevent further potential sequelae . The retrospective nature of this case report is a limitation . Whenever there is a question regarding the etiology of an infectious keratitis, we recommend laboratory analysis to support the clinical diagnosis of hsv keratitis and rule out similar herpes zoster virus keratitis . The prompt response to antiviral therapy supports a herpes virus as the cause of keratitis in these cases but does not exclude the possibility of herpes zoster keratitis . In conclusion, cxl with riboflavin and uv - a can induce herpes keratitis with or without iritis . Prophylactic systemic antiviral treatment in patients with a history of the herpetic disease after cxl with uv - a might decrease the possibility of recurrence.
The influence of culture on the development of eating disorders such as anorexia nervosa (an) and bulimia nervosa (bn) has been long appreciated . These syndromes are more prevalent in industrialized, and often western cultures and are far more common among females than males, mirroring cross - cultural differences in the importance of thinness for women . These patterns indict current cultural beauty ideals in the etiology and maintenance of eating disorders . A secular trend of increased prevalence of bn is observed in the west during the current century, and the recent point prevalence of bn is around 1% of young western women . With another 3 - 5% suffering from similar eating disorders, known as eating disorder not otherwise specified (ednos) in the diagnostic and statistical manual - iv (dsm - iv). Only few classical cases has been reported so far from the asian countries, particularly having more western influence, such as japan, hongkong . Atypical case of bn has recently been reported from india . To our best knowledge, a 22-year - old, unmarried female medical undergraduate, belonging to an urban hindu extended nuclear family of the upper socio - economic status from a metropolitan city, with predominantly narcissistic and a histrionic traits and family history of recurrent depressive disorder in paternal grandmother, presented with the poor eating habits of insidious onset for 9 years . During her 8 class, she developed liking for a boy in her class who rejected her calling fat . Though, she managed to move on; however, developed dissatisfaction for her body image, and would consider herself fat on the mirror and started looking for means to reduce weight . With gradually increasing concern over growing fat, she started skipping two meals and would take only one meal and salads in class 10 . Over next 6 - 7 month period, she lost up to 12 kg and looked thin, although she would consider it inadequate and would find herself flabby, in front of the mirror, although at other times, she could appreciate that her clothes had become loose . However, she never had symptoms of micronutrient deficiency or menstrual irregularity . At the same time, she also developed intense liking for the high calorie foods . She would binge on them 5 - 6 times a month and would regret afterwards . She started exercising for 1 - 1 h in order to compensate weight gain out of binging . This pattern continued for next 1 - 1 when she gave up working out unwillingly, to focus more on studies, and she gained about 4 - 5 kg . She restarted dieting; however, within few months she again started having increased craving for the high calorie foods and binging, which would be more when she would deny food in parties . Though she knew that her body mass index (bmi) was well within normal range, she started taking one tablet of orlistat daily secretly along with skipping meals and rejoining gymnasium in order to reduce her weight to below 50 kg, which was below normal for her height . She could undergo a single session after which it came to the knowledge of a family member, who refrained her . During last 5 year, she would compare herself with every female she met or read about in novels, would feel better on seeing obese females, and feel let down if they were slim ., she would avoid parties, going out with friends, standing for photos, and would spend hours in the gymnasium . At the time of consultation in the psychiatry out - patient department, her bmi was 23, which is within normal range . Her laboratory investigations including, complete hemogram, liver, and renal function tests, serum electrolytes, plasma blood glucose levels were normal . She is under regular opd follow - up with sustained improvement since last 18 weeks . This case is a typical case of bn with obvious presence of a morbid dread of fatness, body image dissatisfaction and setting a sharply defined weight threshold and binging associated with compensatory behavior . Rapid and sustained improvement with the low - dose fluoxetine and cognitive behavioral therapy as observed in this case is usually not seen . Despite ongoing adoption of western values world - wide, body dissatisfaction is remarkably lower in non - western countries . Study on indian medical students by the srinivasan et al . Found 15% of the 210 students had a form of distress and disorder in attitude towards eating habits and body weight, which are milder or subtle than an or bn . The author termed this as eating distress syndrome . Pary - jones, referring to the historical evolution of eating disorder have mentioned about its archaic form, a less sever and benign form of an or bn . The authors stated that the current severe form of eating disorder such as an, bn might have emerged form of this archaic form . This historical evolution of major eating disorder from older form had been observed in studies carried out across different culture and region over different periods of time . Hence, it is possible that major eating disorder might be in evolution phase in countries like india, and largely present here in its archaic form . However, this case may be taken as an indicator of emergence of bn in the context of rapidly increasing western influence in india.
Preterm birth is due to several causes, among which a preexisting occasionally asymptomatic intrauterine infection relatively early in pregnancy should be considered [13]. Thus, amniotic fluid microbial invasion [1, 2, 4, 5] and/or elevated levels of proinflammatory cytokines, chemokines, or other implicated molecules [3, 4, 6, 7] should be investigated . Elastase, a protease produced by neutrophils, histiocytes, and macrophages, is stored in cytoplasmic granules and is secreted during cell activation . It targets at the degradation of intra- or extracellular proteins, among which elastin, collagen, and fibronectin [8, 9] are included . Main inhibitor of elastase is the secretory leukocyte proteinase inhibitor (slpi), present in the secretions of the respiratory and genital system [1012]. It has been shown that slpi limits the proinflammatory cascades ongoing during parturition, protects against microbial invasion and the response to infection, and inhibits the proinflammatory action of bacterial products, for instance of lipopolysaccharides [14, 15]. In general, protease inhibitors, by controlling extracellular matrix proteolysis, contribute to tissue homeostasis . Increased elastase concentrations have been documented at the site of ruptured membranes in cases of preterm delivery . The ratio of elastase to spli concentrations is important for the evolution of a normal delivery, as spli seems to protect both fetal membranes and cervical tissue . Adhesion molecules (soluble intercellular adhesion molecule (sicam-1) and soluble vascular cell adhesion molecule (svcam-1)) are members of the cell - surface immunoglobulin superfamily of adhesion receptors expressed on haematopoietic and nonhaematopoietic cell surfaces, particularly on endothelial cells and induced or upregulated by proinflammatory cytokines (eg, interleukin-1, tumor necrosis factor, interferon-) [20, 21]. As they mediate the adhesion of lymphocytes, monocytes, and eosinophils on activated endothelium, they enable circulating white cells to enter inflamed tissues [22, 23], and thus they are used as markers of inflammation or tissue damage . Both molecules exist in transmembrane and soluble (s) forms [25, 26]. This study was based on the hypothesis that elevated midtrimester amniotic fluid concentrations of elastase, sicam-1, svcam-1, and decreased levels of slpi (all four substances are implicated in the inflammatory process) could possibly serve as useful predictors of asymptomatic intra - amniotic inflammation and/or infection, eventually resulting in preterm labor and delivery . Therefore, we aimed to determine the above substances in the amniotic fluid of women, undergoing second trimester amniocentesis and subsequently delivering pre- or full - term infants . Three hundred and twelve women at the second trimester of pregnancy underwent ultrasound guided transabdominal amniocentesis for several reasons (advanced maternal age, nuchal translucency of the fetus, family history of congenital anomalies, parental hemoglobinopathies). These women belonged to a low - risk pregnancy group, as stated by their private obstetricians, who followed them on a regular basis . Out of the total 312 women, 13 subsequently progressed to spontaneous preterm delivery before 37 weeks of gestation, 6 with and 7 without rupture of membranes . The above 13 women were matched for maternal age, parity, and gestational age at amniocentesis (within 2 weeks) with all eligible controls (21 out of the initial 312 women), who delivered at 37 weeks of gestation or later healthy, appropriate for gestational age neonates (all with birth weights between the 30th and 70th customized centile - controlling for maternal height, booking weight, ethnic group, parity, gestational age, birth weight, and neonatal gender). Premature rupture of membranes, defined as leaking of amniotic fluid before the onset of labor, was absent in 12 and present in 9 out of these 21 controls with term delivery . Women with multiple pregnancy, cervical dilatation (> 1 cm, or ruptured membranes at the time of amniocentesis, abnormal fetal karyotype, or major fetal anomalies were excluded . All included in this study cases and controls were nonsmokers and did not report a previous preterm delivery . Neither clinical signs of chorioamnionitis (temperature 37.8c, uterine tenderness, malodorous vaginal discharge, fetal tachycardia> 160 beats / min, maternal tachycardia> 100 beats / min, and maternal leucocytosis> 15000 cells / mm) nor bleeding during pregnancy was reported . The ethical committee of our teaching hospital approved the collection and the use of these samples . Written informed consent drawn amniotic fluid was centrifuged and stored in polypropylene tubes at 80c until assay . Levels of all substances were determined by commercially available enzyme - linked immunosorbent assays: polymorphonuclear (pmn) elastase, by immundiagnostik ag (d-64625, bensheim), human slpi, human sicam-1, and human svcam-1, r&d systems inc . Sensitivity, intra- and interassay coefficients of variation for pmn elastase were <0.12 ng / ml, 7.5% and 8.4%; for spli <25 pg / ml, 4.5% and 6.2%; for sicam-1 0.35 ng / ml, 3.5% and 5.9%; and for svcam-1 2 ng / ml, 6.3% and 8.2%, respectively . As data from all four substances were normally distributed (kolmogorov - smirnov test), t test was applied for the comparison of investigated amniotic fluid substances between pre- and full - term pregnancies . Nonparametric tests were applied for the comparisons between intact and ruptured membranes in each group . P <.05 was considered statistically significant . A receiver - operating characteristic (roc) curve was used to identify cutoff concentrations of amniotic fluid elastase, spli, sicam-1, and svcam-1 for spontaneous preterm delivery after midtrimester amniocentesis . Pmn elastase, sicam-1, and svcam-1 levels were higher and slpi levels were lower in second - trimester amniotic fluid of mothers delivering preterm as compared to mothers delivering at term, however, these findings did not reach statistical significance . In contrast, slpi levels in second - trimester amniotic fluid were significantly higher in the group of women who delivered either preterm (p <.007) or at term (p <.001) with absence of ruptured membranes prior to delivery (see figure 1). Furthermore, a statistical significant correlation existed between elastase and spli (r = 0.508, p <.002). Roc curve analysis of delivery at <37 weeks for various cutoff levels of elastase, slpi, sicam-1, and svcam-1 was performed . The best cutoff point for elastase was a concentration of 5.72 ng / ml (sensitivity 53.8%, specificity 57.14%, odds ratio (or) 1.6, 95% confidence interval (ci) = 0.4 - 6.3), for spli a concentration of 56.5 ng / ml (sensitivity 53.8%, specificity 42.86%, or = 0.9, 95% ci = 0.2 - 3.5), for sicam-1 a concentration of 116 ng / ml, (sensitivity 62%, specificity 62%, or 2.6, 95% ci: 0.6 - 10.8), and for svcam-1 a concentration of 290 g / dl, (sensitivity 76.9%, specificity 57.1%, or 4.4, 95% ci = 0.9 - 21). In this study we prospectively determined midtrimester amniotic fluid concentrations of several factors and related their levels with pregnancy outcome . Our results indicate that even from the early second trimester of pregnancy, in preterm or full - term deliveries, preceded by rupture of membranes, levels of slpi are significantly decreased, possibly implying influence of the latter on membrane integrity . Previous studies have reported that amniotic fluid protease inhibitors [alpha 1-protease inhibitor (1 -pi), urinary trypsine inhibitor, and slpi] control elastase activity [12, 13, 28, 29]. In this respect, amniotic fluid concentrations of 1 -pi have been found lower in women with rupture of membranes . In addition, zhang et al reported that slpi functions as a potent anti - inflammatory agent by interfering with the signal transduction pathway leading to production of monocyte matrix metalloproteinases, which are also implicated in membrane rupture . On the other hand, the adverse effects of elastase on the growth and properties of elastic tissue in the amnion of rabbits have been demonstrated . Relatively, immunohistochemical studies of fetal ruptured membranes have shown accumulation of elastase at the ruptured site both in full- and preterm deliveries [32, 33]. Amniotic fluid neutrophils are of fetal origin and accumulation of elastase in the amniotic fluid could reflect fetal inflammatory response, as it happens with respective increase of metalloproteinase 8 . A possible explanation for the lower slpi concentrations in cases of ruptured membranes is its consumption early in pregnancy during repeated inflammatory processes . On the other hand, the determination in the amniotic fluid of elastase both in pre- and full - term delivery could imply that this substance is part of the common metabolic pathway of labour, and therefore its concentrations did not change significantly in both groups of the study . Concerning adhesion molecules, previous studies have shown that increased circulating sicam-1 levels in midtrimester amniotic fluid are related to a shortened length of gestation at delivery, that intercellular adhesion molecule-1 concentration, in utero, decreases after antibiotic treatment and that determination of sicam-1, expressed on fetal membranes and mononuclear cells of amniotic fluid, may be a valuable biomarker for early detection of acute chorioamnionitis and the possibility of premature rupture of membranes . Nevertheless, another study states that in contrast to other proinflammatory molecules (interleukin-6 and leukocyte adhesion molecule-1), amniotic fluid sicam-1 concentrations were not significantly different between patients with intra - amniotic infection than without intra - amniotic infection, a finding being in accordance with the relevant result of the present study, referring to incidence of preterm delivery . Lastly, to the best of our knowledge, no study could be found determining amniotic fluid concentrations of svcam-1 in midtrimester . In conclusion, second - trimester amniotic fluid slpi levels are significantly decreased in cases where pre- or full - term delivery is preceded by membrane rupture . Therefore, slpi concentrations in the amniotic fluid obtained by second - trimester amniocentesis could possibly predict the rupture of membranes either in the second or in the third trimester . In contrast, based on this study, midtrimester amniotic fluid elastase, slpi, sicam-1, and svcam-1 concentrations are not helpful in predicting preterm delivery . Levels of slpi in women, who delivered either preterm (a) or at term (b) with intact membranes (a) n = 7 and (b) n = 12 or premature rupture of membranes (prom) (a) n = 6 and (b) n = 9, respectively . Demographic data of participating women delivering at <37 weeks of gestation (cases) or at term (controls) (mean sd).
The prevalence of diabetes mellitus and suboptimal achievement of glycaemic control is increasing in asian populations.1, 2, 3 evidence shows that there may be differences in the pathophysiology of type 2 diabetes across ethnic subgroups, with asian vs nonasian patients exhibiting a propensity for lower body mass index, a higher proportion of visceral fat and predominant insulin secretory defects.4, 5 although pharmacologic agents are available to patients with type 2 diabetes who fail to achieve sufficient glycaemic control through diet and exercise, many of these agents may cause hypoglycaemia or weight gain.6, 7 these adverse events (aes) often negatively affect treatment adherence and/or longterm use . Therefore, there is a need for effective, longterm glycaemic control regimens that can be administered safely . Alogliptin is a potent, highly selective, orally available dipeptidyl peptidase4 (dpp4) inhibitor . Since 2013, alogliptin has been available in the usa and the european union as monotherapy and as a fixeddose combination (fdc) with metformin . Mechanistically, inhibition of dpp4 activity leads to an increase in hormone glucagonlike peptide 1 levels, which in turn stimulates glucosedependent insulin secretion from pancreatic cells.8 this mechanism of achieving glucose homeostasis is distinct from that of metformin, which is a biguanide that suppresses hepatic glucose release and also induces weight loss via reduced calorie intake.9 given the complementary mechanisms of action of alogliptin and metformin, and the potential for incrementally improving glycaemic control without exacerbating safety events, there has been interest in the use of this fdc in patients with type 2 diabetes . Phase 3 clinical trials in asian patients have shown that concomitant administration of alogliptin and metformin is effective and has a safety profile similar to that of the individual components.10, 11 the current phase 3 study was designed to evaluate the efficacy and safety of alogliptin 12.5 mg plus metformin 500 mg fdc bid vs alogliptin or metformin alone in asian patients with type 2 diabetes who are inadequately controlled with diet and exercise . This was a phase 3, randomized, doubleblind, placebocontrolled, multicentre study that was conducted at 59 sites in china, malaysia, republic of korea (south korea) and taiwan . It was conducted in compliance with the study protocol, the ethical principles originating in the declaration of helsinki and the international conference on harmonisation of tripartite guidelines for good clinical practice . Patients aged 18 to 75 years were eligible if they had an historical diagnosis of type 2 diabetes for which glycaemic control was inadequate (ie, hba1c of 7.5%10.0% after at least 2 months of diet and exercise prior to the screening period). The study consisted of 4 periods: screening (2 weeks), placebo runin (4 weeks), treatment (26 weeks) and followup (2 weeks). Eligible patients had hba1c of 7.5% to 10.0% and fasting plasma glucose (fpg) 275 mg / dl (15.27 mmol / l) at the week 1 visit of the placebo runin (1 week prior to the treatment phase). After stratifying patients by screening hba1c (<8.5% vs 8.5%) and by country, patients were randomized 1:1:1:1: to receive placebo, alogliptin 12.5 mg twicedaily (bid), metformin 500 mg bid or fdc of alogliptin 12.5 mg bid plus metformin 500 mg bid . The primary efficacy variable was change from baseline in hba1c at week 26 (or early termination) using last observation carried forward . For this variable, 130 patients per treatment group ensured at least 90% power to demonstrate that alogliptin + metformin fdc bid was statistically superior to constituent doses of alogliptin and metformin . This power calculation assumed a treatment effect of 0.45% between the fdc and constituent doses . The primary efficacy analysis was conducted using the full analysis set (fas) and an analysis of covariance (ancova) model, with change from baseline in hba1c at week 26 (or early termination) as the response variable . Treatment and country were fixed effects and baseline hba1c was a continuous covariate (model 1). A supportive analysis was also conducted using model 1 and the perprotocol analysis set (pps; ie, patients in the fas without major protocol deviations). Changes from baseline in hba1c and fpg (leastsquares [ls] means) were summarized at each time point using descriptive statistics . The incidences of hyperglycaemic rescue (for fpg 275 mg / dl [15.27 mmol / l] before week 12 or hba1c 8.5% and 0.5% reduction in hba1c between week 12 and the final visit), marked hyperglycaemia (fpg> 200 mg / dl [11.1 mmol / l]) and clinical response were also summarized (frequency and percentage) by treatment group . Continuous secondary endpoints were analysed using an appropriate statistical model with the model 1 variables . The analysis was carried out using a cox proportional hazard model, with treatment as an effect, country as a stratification factor and baseline hba1c as a covariate . For each efficacy variable, the corresponding baseline value for that variable was modeled as a covariate in place of baseline hba1c . Treatmentemergent aes (including all events, irrespective of treatment relationship and treatmentrelated aes), clinical laboratory changes and hypoglycaemic episodes were summarized . Of 1258 screened patients, 647 were randomized to placebo (n = 163), alogliptin (n = 163), metformin (n = 162) or alogliptin + metformin fdc (n = 159) and were included in the efficacy and safety populations (figure s1, file s1). Demographic and clinical characteristics for the randomized population were similar between study groups (table s1, file s1). Of the 647 treated patients, 511 (n = 104, placebo; n = 126, alogliptin; n = 135, metformin; n = 146, alogliptin + metformin fdc) completed the study . The most common reason for study discontinuation was lack of efficacy (n = 86 [64.2%] all groups). The ls mean change in hba1c from baseline to end of treatment (week 26) was 0.19% with placebo, 0.86% with alogliptin, 1.04% with metformin and 1.53% with alogliptin + metformin bid (table 1). Changes were significantly greater (p <.0001) for alogliptin + metformin fdc vs metformin alone (ls mean difference 0.49%; 95% confidence interval [ci] 0.700, 0.278) and alogliptin alone (ls mean difference 0.68%; 95% ci 0.889, 0.467). Similar results were observed in a supportive analysis using the pps: alogliptin + metformin fdc bid led to significantly (p <.0001) greater reductions from baseline in hba1c at week 26 compared with metformin bid or alogliptin bid alone (ls mean differences of 0.49% and 0.66%, respectively). Significantly greater decreases in mean hba1c and fpg were observed with alogliptin + metformin fdc vs either alogliptin or metformin alone, beginning with the first ontreatment assessment at week 4 and continuing for all time points throughout the study (p <.02 for hba1c, for all comparisons; p .002 for fpg, for all comparisons) (figure 1). Change from baseline in hba1c (%) to week 26 abbreviations: ci, confidence interval; hba1c, haemoglobin a1c; ls, leastsquares; sd, standard deviation; se, standard error . Covariates were treatment, country, baseline hba1c, treatment by baseline hba1c and treatment by country interaction . Analysis of adjusted mean change from baseline in (a) haemoglobin a1c (hba1c;%) and (b) fasting plasma glucose (fpg; mmol / l) to week 26 . P <.02 for hba1c, for all comparisons; p .002 for fpg, for all comparisons . Bid, twice daily; fdc, fixeddose combination . A lower proportion of patients in the alogliptin + metformin fdc group required hyperglycaemic rescue by week 26 (4.4%) compared with the proportion of patients in the placebo group (25.5%), alogliptin (14.8%) and metformin (8.7%) groups; this difference was significant as compared with the alogliptin group (p = .002). A similar trend was observed for marked hyperglycaemia (p = .040 vs alogliptin). For time to hyperglycaemic rescue, both placebo and metformin had a significantly lower adjusted risk ratio vs alogliptin + metformin fdc (p <.0001 and p = .002, respectively) (figure s2, file s1). A higher proportion of patients in the alogliptin + metformin fdc group had hba1c clinical response at week 26 as compared with patients in the other 3 treatment groups (table s2, file s1). The overall frequency of treatmentemergent aes was similar for the 4 treatment groups: 48.4% for placebo, 46.3% for alogliptin, 47.2% for metformin and 45.6% for alogliptin + metformin fdc (table s3, file s1). Across all treatment groups, upper respiratory tract infection was the most common ae, and the only ae with 5% incidence (range 4.9%6.8%). Two saes were considered to be related to treatment: moderate gastroenteritis (1 event; placebo) that resolved in 8 days, and moderate unstable angina (1 event; alogliptin + metformin fdc) that resolved in 9 days . Overall, 19 patients (3.0%) experienced 23 hypoglycaemic events during the study, 6.2% in the metformin group, 3.8% in the alogliptin + metformin fdc group, 1.2% in the alogliptin group and 0.6% in the placebo group . The ls mean change in hba1c from baseline to end of treatment (week 26) was 0.19% with placebo, 0.86% with alogliptin, 1.04% with metformin and 1.53% with alogliptin + metformin bid (table 1). Changes were significantly greater (p <.0001) for alogliptin + metformin fdc vs metformin alone (ls mean difference 0.49%; 95% confidence interval [ci] 0.700, 0.278) and alogliptin alone (ls mean difference 0.68%; 95% ci 0.889, 0.467). Similar results were observed in a supportive analysis using the pps: alogliptin + metformin fdc bid led to significantly (p <.0001) greater reductions from baseline in hba1c at week 26 compared with metformin bid or alogliptin bid alone (ls mean differences of 0.49% and 0.66%, respectively). Significantly greater decreases in mean hba1c and fpg were observed with alogliptin + metformin fdc vs either alogliptin or metformin alone, beginning with the first ontreatment assessment at week 4 and continuing for all time points throughout the study (p <.02 for hba1c, for all comparisons; p .002 for fpg, for all comparisons) (figure 1). Change from baseline in hba1c (%) to week 26 abbreviations: ci, confidence interval; hba1c, haemoglobin a1c; ls, leastsquares; sd, standard deviation; se, standard error . Covariates were treatment, country, baseline hba1c, treatment by baseline hba1c and treatment by country interaction . Analysis of adjusted mean change from baseline in (a) haemoglobin a1c (hba1c;%) and (b) fasting plasma glucose (fpg; mmol / l) to week 26 . P <.02 for hba1c, for all comparisons; p .002 for fpg, for all comparisons . . A lower proportion of patients in the alogliptin + metformin fdc group required hyperglycaemic rescue by week 26 (4.4%) compared with the proportion of patients in the placebo group (25.5%), alogliptin (14.8%) and metformin (8.7%) groups; this difference was significant as compared with the alogliptin group (p = .002). A similar trend was observed for marked hyperglycaemia (p = .040 vs alogliptin). For time to hyperglycaemic rescue, both placebo and metformin had a significantly lower adjusted risk ratio vs alogliptin + metformin fdc (p <.0001 and p = .002, respectively) (figure s2, file s1). A higher proportion of patients in the alogliptin + metformin fdc group had hba1c clinical response at week 26 as compared with patients in the other 3 treatment groups (table s2, file s1). The overall frequency of treatmentemergent aes was similar for the 4 treatment groups: 48.4% for placebo, 46.3% for alogliptin, 47.2% for metformin and 45.6% for alogliptin + metformin fdc (table s3, file s1). Across all treatment groups, upper respiratory tract infection was the most common ae, and the only ae with 5% incidence (range 4.9%6.8%). Two saes were considered to be related to treatment: moderate gastroenteritis (1 event; placebo) that resolved in 8 days, and moderate unstable angina (1 event; alogliptin + metformin fdc) that resolved in 9 days . Overall, 19 patients (3.0%) experienced 23 hypoglycaemic events during the study, 6.2% in the metformin group, 3.8% in the alogliptin + metformin fdc group, 1.2% in the alogliptin group and 0.6% in the placebo group . This phase 3 study demonstrated significantly greater reductions in hba1c over 26 weeks with alogliptin + metformin fdc vs alogliptin or metformin . The advantages of the fdc regimen in reducing hba1c (primary endpoint) and fpg (secondary endpoint) were evident at week 4, with sustained effects throughout the study . By week 26, the rate of achieving clinical response measures was higher and the rate of hyperglycaemic rescue or marked hyperglycaemia was lower with alogliptin + metformin fdc . The incidence of hypoglycaemia (6.2%) was highest in the metforminalone group, which is difficult to explain, as the incidence of hypoglycaemia in the alogliptin + metformin fdc group was lower (3.8%). The overall incidence of aes and saes did not differ with alogliptin + metformin fdc vs each monotherapy . This finding is consistent with data from prior phase 3 clinical trials that tested concomitant alogliptin + metformin in asian patients.10, 11, 12 in addition, a metaanalysis of various ddp4 inhibitors plus metformin as initial therapy revealed no increase in the risk of hypoglycaemia or prolonged gastrointestinal complaints relative to metformin monotherapy.13 the efficacy and safety of alogliptin + metformin fdc demonstrated in this study is of particular importance because of potential differences in the pathophysiology of type 2 diabetes in asians compared with other populations . Furthermore, there are multiple reasons why alogliptin + metformin fdc represents an important addition to the glycaemiclowering armamentarium in asia . First, patients are predisposed to multiple comorbidities and polypharmacy, and improving medication adherence is associated with better glycaemic control.14 second, the fdc incorporates 2 commonly prescribed classes of a type 2 diabetes agent with different mechanisms of action . Metformin is recommended in international diabetes guidelines as firstline treatment of patients with type 2 diabetes,15 while dpp4 inhibitors are recommended in the secondline setting . Similar recommendations have been made in asian diabetes guidelines, such as those published by the chinese diabetes society.16 last, the endure study, where alogliptin was added to stable doses of metformin, demonstrated the sustainability of glycaemic control.17 one limitation of the study is its relatively short duration, which provided no insight into the durability of hba1c lowering, longterm tolerability or adherence . However, the durability of the treatment effect was addressed in the endure study.17 additionally, our study was conducted in eastern asian countries and employed extensive eligibility criteria, which precludes the extrapolation of results to the entire type 2 diabetes population . In conclusion, alogliptin + metformin fdc bid resulted in better glycaemic control as compared with alogliptin or metformin alone in asian patients with type 2 diabetes who were inadequately controlled with diet and exercise . Eligibility criteria, patient demographic and clinical characteristics, incidence of hba1c clinical response at week 26, adverse events, patient disposition, and time to hyperglycemic rescue click here for additional data file.
In 1939, sigurjonsson reported that the thyroid gland in the icelandic population appeared to be very small compared to the commonly accepted normal size in other countries (11). The first study on nutrition and diet of icelanders showed that fish as well as dairy products were the main iodine sources (12). The high consumption of fish, 200 g / day on average, was considered unique compared to other countries (table 1). Milk and dairy product consumption was also high, on an average 1,000 ml / day . Considering that 100 g of fish provides on an average 180 g / day (6) and 100 ml of milk around 21 g / day in this period (9), approximately 570 g of iodine was provided daily through consumption of these products (table 1). This level of consumption is close to the upper safe limit of iodine intake (600 g / day) proposed by the scientific committee on food (13). However, persons with normal thyroid function can in general tolerate a prolonged consumption of iodine up to 1 mg / day (14), suggesting that the high iodine consumption of the icelandic population probably did not cause any harm . Intake of fish, milk, and dairy products in icelandic dietary surveys, and estimated iodine intake estimates of sd not available . Estimated iodine intake based in information on intake of fish (estimated iodine content of 191 g/100 g) and milk (estimated iodine content of 21 g/100 g). Figures are estimates as the intake was presented as percentages of total energy intake in the report presenting the data . A second diet survey was performed in 19791980 by the icelandic nutrition council (15). In the council's report presenting the main results of the study, the food group fish, meat and eggs provided 20% of the energy intake . The average energy intake of women was 1,970 kcal / day and for men 2,750 kcal / day, meaning that fish, meat, and eggs provided about 400550 kcal per day . Food supply data suggest the proportional intake of meat, fish, and eggs in 1979 was very similar to that in year 2002 (7, 16). Using this assumption, about 15% of the total energy from the food group (fish, meat, and eggs) is attributable to fish, and the average fish intake could be estimated as around 70 g / day (60 g / day for women and 80 g / day for men), given that 100 g provide around 100 kcal (6) (table 1). In the 19791980 study, milk and dairy products provided 21% of the total energy . Given that most of the milk consumed was whole milk providing 68 kcal/100 g, the estimated average intake of milk and dairy products is 729 g / day . This level of intake harmonizes very well with data on food supplies in iceland in 1979, was estimated to be 269 kg / person / year or 737 g / day (16). Interestingly, the authors of the report 1979 mentioned changes in diet habits, especially by the younger generation and a risk of nutritional deficiency due to poor diet (15). The average intake of iodine was estimated to be 336 g / day, using the same assumptions as in the previous section (only based on intake of fish, milk, and dairy products). In 1988 a second study of iodine status on icelanders was performed, including 2059 years old individuals showing excellent iodine status with average iodine excretion of 395 g / l for men and 270 g / l for women (9). In 1990, the icelandic nutrition council conducted a third national dietary survey (8). The average fish consumption was 73 g / day, which was a huge drop from the survey in 1939, although similar to the estimated intake in 19791980 (table 1). People above 50 years had the highest fish consumption but the younger generations consumed considerably less . The average consumption of milk was 320 g / day but on an average the total consumption of dairy products was 589 g / day (table 1). Although the intake of fish and milk had decreased, the average intake of iodine was 299 g / day, 365 g / day for men and 238 g / day for women (table 1). However, the study revealed a group of young women, consuming on an average less than 22 g fish per day, and very low amount of dairy products as well . The average intake of iodine by these young women ranged from 86 g / day to 130 g / day which is below the rdi of iodine . These results were the first evidences to reveal a certain group in iceland which could be at risk for iodine deficiency (8). In 1998 the iodine concentration was measured in the urine of 6670 years old danes and icelanders (17). The results showed that the iodine status of icelanders was still optimal, on an average150 g / l, ranging from 33 g / l to 703 g / l . However, it was lower than in the previous studies, indicating changes in dietary habits . It should be noted, however, that this study only included elderly individuals and not the group of young women suspected to be at risk of iodine deficiency in the dietary survey 1990 (8). Data collection is ongoing to study further iodine status of young women (1620 years old) and pregnant women in iceland, where both iodine intake and urinary iodine excretion will be estimated . The latest dietary survey was performed by the icelandic public health institute (formerly nutrition coucil) in 2002 and the results showed quite extensive changes in food consumption, compared with the survey in 1990 (7, 8). As in 1990 the intake of fish was split by generations, the younger people consumed three times less fish than the older people and the young women consumed the smallest serving of fish of all participants, only 15 g / day, and 23% of young women (1519 years) consumed fish less than once a week . Milk consumption was also considerably lower than before, 185 g / day on average . However, the average consumption of other dairy products seemed to have increased resulting in an average intake of milk and dairy products corresponding to 388 g / day (table 1). Those consuming least dairy were young women (1519 years), where 17% consumed dairy products less than once a day . The average iodine intake by men was 196 g / day and by women 135 g / day . On average, young women (1519 years) only obtained 2/3 of rdi for iodine from the diet . Similar trends presumably due to reduction of fish intake and milk consumption have been seen in studies on children and adolescents in iceland where up to 44% of 15-year - old adolescents were estimated to be at risk of iodine deficiency due to low intake of fish and dairy products (18). Very low fish intake has also been seen in young overweight icelanders (19). Both dietary intake and composition of food is in continuous changes, and an example is icelandic milk where the iodine concentration has decreased by 48% from 1962 to 1997 (6, 9). Milk intake has also decreased in the same period (7, 8, 12, 15). Therefore, follow - up studies are essential to evaluate dietary intake and nutrient status on regular basis, and in iceland a study on iodine status of young women is essential . The latest dietary survey, from 2002, showed the diet to have become more similar to the diet in other european countries and the consumption of fish and dairy products had diminished significantly . These changes were seen most clearly for young people, but previously the references for iodine status in iceland had been considered outstanding due to the good iodine status in earlier times (18). According to the latest diet research, intake of salt is too high in iceland, like in other western countries (7). Although availability of iodized salt would not necessarily result in increased salt consumption, this solution could easily be criticized . The official icelandic recommended intake of fish is 300 g / week and two portions of milk or dairy products per day . By following these recommendations around 138 g iodine a group of young icelandic women in childbearing age might be at risk of iodine deficiency . The seriousness of the matter is that in the next few years these women might get pregnant where an iodine deficiency could have some negative consequences for the future generations due to its effects on the maturation of the fetuses and neonates by irreversible derangement in the development of the brain and central nervous system (20, 21). Some but not all dietary supplements sold in iceland contain iodine and emphasis should be made on getting vitamins and minerals from food . Fish and milk are the foods which are recommended because of the content of other important nutrients . In conclusion, trends in dietary intake in iceland show decreased fish and dairy intake especially by the young generation . The results from the ongoing studies on iodine status of icelandic adolescent girls and pregnant women are important to evaluate the need for special action with regard to iodine status . The author has not received any funding or benefits from industry to conduct this study.
Acute dorsolateral striatum slices were prepared from p58p87 hemizygous bacterial artificial chromosome (bac) d2 dopamine receptor - egfp (d2-egfp) or d1 dopamine receptor - tdtomato (d1-tdtomato) mice, males and females in the c57bl/6 background . In accordance with northwestern university animal studies committee, mice were deeply anesthetized with ketamine and xylazine, perfused intracardially with ice - cold modified artificial cerebrospinal fluid (acsf) (in mm): 125 nacl, 7 glucose, 25 nahco3, 2.5 kcl, 1.25 nah2po4, equilibrated with 95% oxygen, 5% co2 plus 0.5 mm cacl2, 2 mm mgcl2, 2 mm ascorbic acid . Coronal slices, 250 m, were cut in the above modified acsf, then incubated at 32c for 30 min in the above modified acsf containing 2 mm cacl2, 1 mm mgcl2, 5 mm l - glutathione and 1 m pyruvate and subsequently stored at room temperature . Except when noted, drugs were obtained from sigma, tocris, or invitrogen . Slices were perfused at 23 ml per minute with oxygenated modified acsf containing 2 mm cacl2 and 1 mm mgcl2 at 25c . For all experiments, 20 m gabazine and 2 m cgp55845 were added to the perfusion to block gabaa and gabab receptors, respectively . Spns restricted to the dorsolateral striatum were identified using infrared differential interference contrast on an upright olympus microscope and a cooled ccd camera (coolsnap hq) controlled with metamorph software . In bac d1-tdtomato mice, indirect pathway spns in these mice were identified by the lack of fluorescence in the soma, possessing spines (visualized with the aid of an alexa fluor hydrazide fluorescent dye), and passive membrane properties of spns . In bac d2-egfp mice, direct pathway spns in these mice were identified by the lack of fluorescence, possessing spines (visualized with the aid of an alexa fluor hydrazide fluorescent dye), and passive membrane properties of spns . Dual simultaneous somatic whole - cell recordings were made with borosilicate patch pipettes having open tip resistances of 34.5 mohms . The intracellular pipette solution contained (in mm): 115 cesium methylsulfonate, 5 hepes, 5 tetraethyammonium - cl, 2 qx-314, 0.25 egta, 2 mg - atp, 0.5 na - gtp, 10 sodium phosphocreatine, and 100200 m alexa fluor 488 or 568 hydrazide, ph 7.3 and osmolarity 290 . After dual somatic recordings were established for a randomly selected dspn and ispn pair, no more than 70 m apart, with similar depths from the slice surface (cell body of each pair had no more than 10 m depth difference between each other), the cells were allowed to dialyze with the internal solution for 710 mins . Epscs were occluded with bath application of 100 m d-(-)-2-amino-5-phosphonopentanoic acid (apv) and 20 m 6-cyano-7-nitroquinoxaline-2,3-dione (cnqx) or 1 m tetrodotoxin . Data were acquired at 100 khz, filtered at 10 khz using an 8-pole bessel filter, and digitized using a digidata 1440 16-bit a / d converter . Somatic access resistance <25 mohms was monitored and cells with unstable access resistance (> 30% change) were discarded . Cells were held at 80 mv, not corrected for liquid junction potential (calculated to be approximately 11 mv). Stereotaxic guided surgeries were performed on the above mice anesthetized with ketamine and xylazine mixture or isoflurane . After positioning the head to obtain a flat skull between bregma and lamda, a small hole was bored with a micro drill bit, and a glass pipette was slowly inserted at the following coordinates . Dorsolateral striatum injection coordinates (relative to bregma) were: 2.0 mm posterior, 3.5 mm lateral, and 3.5 mm ventral; with the manipulator tiled 48 from the z - axis and 72 from the y - axis . Pons injection coordinates (relative to lambda) were: 2.0 and 2.5 mm posterior, 0.5 mm lateral, and 5.56.5 mm ventral; with the manipulator tilted 12 from the z - axis, and the nose bar adjusted to 30 from x - y plane . Motor cortex injection coordinates (relative to bregma) were: 1.6 mm anterior, 1.2 mm lateral and 0.75 mm ventral . To minimize diffusion, over the course of 1 minute the following volumes were slowly injected: 0.2 l of a recombinant rabies virus carrying a channelrhodopsin-2-venus expression construct (rrabies - chr2-vn) for pons, 0.1 l rrabies - chr2-vn for striatum, and 0.1 l of an adeno - associated virus carrying a chr2-venus expression construct (aav2/9-chr2-vn, supplied by u. penn vector core; addgene 20071) for motor cortex . Successful targeting produced fluorescence within the pontine nuclei, dorsolateral striatum, or motor cortex, as well as appropriate placement of fluorescent neurons within the cortex . To activate channelrhodopsin containing axons within the dorsolateral striatum, 473 nm light was generated with a crystalaser (safety protocols for class iiib laser), intensity - modulated with a conoptics pockels cell, expanded with concave lens (thorlabs), passed through an adjustable iris, and 5 objective lens (0.15 na) to produce a 700 m diameter column of light at the slice surface . Blue light pulses (3 ms) were triggered with a digidata interface that gated a fast uniblitz shutter . Light power was calibrated with a fast photo - diode (thorlabs) at the laser and sample . Light intensity used for maximal activation was approximately 25 mw, and 5 mw for submaximal activation (producing at minimum a 20% decrease in epsc amplitude compared to the epsc amplitude at 25 mw). The amplitudes of epscs were averaged from at least 3 trials (up to 10 trials), with an intersweep interval of 1 minute . Images were acquired on an upright olympus or zeiss microscope, qimaging or photometrics camera and with ephus, metamorph, or qcature software . Images were adjusted in nih imagej or adobe photoshop for brightness, contrast, and pseudocoloring . Axon pclamp data files was imported into igor (wavemetrics) and custom written routines were used to analyze the data . Pilot studies to determine the variability of outcome measures and the effective size were used to estimate the number of observations necessary to test the null hypothesis . All graphically representations display the ranks of observations within a group using boxplots, illustrating the median (thick line), interquartile range, and minimum / maximum range whiskers . Reponses smaller than 34 times the root mean square of the baseline noise average were displayed as 0.1 on the log scale plots, for graphical reasons . Data points falling outside of the following range were not included in the analysis: (the interquartile range multiplied by 3 and then subtracted from the lower quartile value) and (the interquartile range multiplied by 3 and then added to the upper quartile value). Epsc peak amplitudes greater than 2 na were not included in the data set due to changes in voltage control . Distribution - free statistical analysis were performed in graphpad prism or originpro, using the nonparametric wilcoxon matched - pairs signed rank two - tailed test or a wilcoxon signed - rank test comparing the ratio medians to 1 . Statistical significance (p <0.05) acute dorsolateral striatum slices were prepared from p58p87 hemizygous bacterial artificial chromosome (bac) d2 dopamine receptor - egfp (d2-egfp) or d1 dopamine receptor - tdtomato (d1-tdtomato) mice, males and females in the c57bl/6 background . In accordance with northwestern university animal studies committee, mice were deeply anesthetized with ketamine and xylazine, perfused intracardially with ice - cold modified artificial cerebrospinal fluid (acsf) (in mm): 125 nacl, 7 glucose, 25 nahco3, 2.5 kcl, 1.25 nah2po4, equilibrated with 95% oxygen, 5% co2 plus 0.5 mm cacl2, 2 mm mgcl2, 2 mm ascorbic acid . Coronal slices, 250 m, were cut in the above modified acsf, then incubated at 32c for 30 min in the above modified acsf containing 2 mm cacl2, 1 mm mgcl2, 5 mm l - glutathione and 1 m pyruvate and subsequently stored at room temperature . Except when noted, drugs were obtained from sigma, tocris, or invitrogen . Slices were perfused at 23 ml per minute with oxygenated modified acsf containing 2 mm cacl2 and 1 mm mgcl2 at 25c . For all experiments, 20 m gabazine and 2 m cgp55845 were added to the perfusion to block gabaa and gabab receptors, respectively . Spns restricted to the dorsolateral striatum were identified using infrared differential interference contrast on an upright olympus microscope and a cooled ccd camera (coolsnap hq) controlled with metamorph software . In bac d1-tdtomato mice, indirect pathway spns in these mice were identified by the lack of fluorescence in the soma, possessing spines (visualized with the aid of an alexa fluor hydrazide fluorescent dye), and passive membrane properties of spns . In bac d2-egfp mice, direct pathway spns in these mice were identified by the lack of fluorescence, possessing spines (visualized with the aid of an alexa fluor hydrazide fluorescent dye), and passive membrane properties of spns . Dual simultaneous somatic whole - cell recordings were made with borosilicate patch pipettes having open tip resistances of 34.5 mohms . The intracellular pipette solution contained (in mm): 115 cesium methylsulfonate, 5 hepes, 5 tetraethyammonium - cl, 2 qx-314, 0.25 egta, 2 mg - atp, 0.5 na - gtp, 10 sodium phosphocreatine, and 100200 m alexa fluor 488 or 568 hydrazide, ph 7.3 and osmolarity 290 . After dual somatic recordings were established for a randomly selected dspn and ispn pair, no more than 70 m apart, with similar depths from the slice surface (cell body of each pair had no more than 10 m depth difference between each other), the cells were allowed to dialyze with the internal solution for 710 mins . Epscs were occluded with bath application of 100 m d-(-)-2-amino-5-phosphonopentanoic acid (apv) and 20 m 6-cyano-7-nitroquinoxaline-2,3-dione (cnqx) or 1 m tetrodotoxin . Data were acquired at 100 khz, filtered at 10 khz using an 8-pole bessel filter, and digitized using a digidata 1440 16-bit a / d converter . Somatic access resistance <25 mohms was monitored and cells with unstable access resistance (> 30% change) were discarded . Cells were held at 80 mv, not corrected for liquid junction potential (calculated to be approximately 11 mv). Stereotaxic guided surgeries were performed on the above mice anesthetized with ketamine and xylazine mixture or isoflurane . After positioning the head to obtain a flat skull between bregma and lamda, a small hole was bored with a micro drill bit, and a glass pipette was slowly inserted at the following coordinates . Dorsolateral striatum injection coordinates (relative to bregma) were: 2.0 mm posterior, 3.5 mm lateral, and 3.5 mm ventral; with the manipulator tiled 48 from the z - axis and 72 from the y - axis . Pons injection coordinates (relative to lambda) were: 2.0 and 2.5 mm posterior, 0.5 mm lateral, and 5.56.5 mm ventral; with the manipulator tilted 12 from the z - axis, and the nose bar adjusted to 30 from x - y plane . Motor cortex injection coordinates (relative to bregma) were: 1.6 mm anterior, 1.2 mm lateral and 0.75 mm ventral . To minimize diffusion, over the course of 1 minute the following volumes were slowly injected: 0.2 l of a recombinant rabies virus carrying a channelrhodopsin-2-venus expression construct (rrabies - chr2-vn) for pons, 0.1 l rrabies - chr2-vn for striatum, and 0.1 l of an adeno - associated virus carrying a chr2-venus expression construct (aav2/9-chr2-vn, supplied by u. penn vector core; addgene 20071) for motor cortex . Successful targeting produced fluorescence within the pontine nuclei, dorsolateral striatum, or motor cortex, as well as appropriate placement of fluorescent neurons within the cortex . To activate channelrhodopsin containing axons within the dorsolateral striatum, 473 nm light was generated with a crystalaser (safety protocols for class iiib laser), intensity - modulated with a conoptics pockels cell, expanded with concave lens (thorlabs), passed through an adjustable iris, and 5 objective lens (0.15 na) to produce a 700 m diameter column of light at the slice surface . Blue light pulses (3 ms) were triggered with a digidata interface that gated a fast uniblitz shutter . Light power was calibrated with a fast photo - diode (thorlabs) at the laser and sample . Light intensity used for maximal activation was approximately 25 mw, and 5 mw for submaximal activation (producing at minimum a 20% decrease in epsc amplitude compared to the epsc amplitude at 25 mw). The amplitudes of epscs were averaged from at least 3 trials (up to 10 trials), with an intersweep interval of 1 minute . Images were acquired on an upright olympus or zeiss microscope, qimaging or photometrics camera and with ephus, metamorph, or qcature software . Images were adjusted in nih imagej or adobe photoshop for brightness, contrast, and pseudocoloring . Axon pclamp data files was imported into igor (wavemetrics) and custom written routines were used to analyze the data . Pilot studies to determine the variability of outcome measures and the effective size were used to estimate the number of observations necessary to test the null hypothesis . All graphically representations display the ranks of observations within a group using boxplots, illustrating the median (thick line), interquartile range, and minimum / maximum range whiskers . Reponses smaller than 34 times the root mean square of the baseline noise average were displayed as 0.1 on the log scale plots, for graphical reasons . Data points falling outside of the following range were not included in the analysis: (the interquartile range multiplied by 3 and then subtracted from the lower quartile value) and (the interquartile range multiplied by 3 and then added to the upper quartile value). Epsc peak amplitudes greater than 2 na were not included in the data set due to changes in voltage control . Distribution - free statistical analysis were performed in graphpad prism or originpro, using the nonparametric wilcoxon matched - pairs signed rank two - tailed test or a wilcoxon signed - rank test comparing the ratio medians to 1.

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