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Medical images are popular in real world because they give intuitive expression . With the development of image processing, computer - aided diagnosis are more important with the ever increasing images . But medical images are often with intensity inhomogeneities; so it is a hard work to segment in such images as angiogram and mr bladder images . The active contour model has been increasingly applied to image segmentation in the past decade, because it provides very good frameworks of variational image segmentations . Chan - vese (cv) model is the most popular active contour model for image segmentation based on the feature of mean gray value of different regions . The variance information can solve the question of different areas with same mean gray value and different variance . The map (maximum a posterior) is an efficient region descriptor for the segmentation task . Zhu, paragios and deriche, and rousson and deriche approximated the map of the given image by a mixture of gaussians . The features deduced by map method are very popular in images with different probability distributions . But they are a failure in image segmentation with intensity inhomogeneities, because there are no features that can be deduced for segmentation using the active contour model . The method is proposed for medical images with complex topological structure, strong contrast, and low noise characteristics . It makes full use of the image area information, builds an energy model, and uses variation gradient information to establish a global energy model to get the minimization value . They combine active contour model with diffusion filter for multiobject segmentation of the noisy image . A target adaptive scheme is designed for adjusting the model to solving a particular image processing task . All the methods mentioned above are region - based models aiming to identify each region of interest by using a certain region descriptor to guide the motion of the active contour . For images with intensity inhomogeneity, there is much important information such as image gradient and laplace that are ignored in active contour model . For medical images, intensity inhomogeneity is usually due to technical limitations or artifacts introduced by the object being imaged . So incorporate edge information alone is not enough, we also incorporate higher order diffusion term into the active contour model aiding segmentation . In this paper, we propose a new active contour model with gradient and higher - order information of the image . The model can also be deemed as active contour model coupling with high - order diffusion, diffusion, because the introduction of laplace information and the difference scheme becomes more difficult . To enhance the efficiency of the segmentation, the fast split bregman algorithm is designed for the segmentation implementation ., we will introduce the active contour and high - order diffusion related methods briefly . Then the new active model with high - order diffusion method is proposed in section 3 . Generalized perona - malik equation for image restoration was the first paper that introduced and implemented high - order anisotropic diffusion partial differential equations (pdes) for image analysis . There are also many other high - order pdes that have been already applied to image processing [13, 14]. Incorporating laplace term, higher - order diffusion can use more information near the edges . In this paper, we select the total variation with higher - order diffusion because of the simplicity . The total variation model with higher - order diffusion is depicted as: (1)e(u)=12(uf)2dx dy+1\|u\|dx dy+2\|u\|dx dy, where is the image domain, u is the denoised image, and f is the noisy image . The active contour model using mean gray value is depicted as: (2)arg min{\|\|dx dy + (1\|fu1\|2+2\|fu2\|2(1))dx dy}, where is the level set function and u 1 and u 2 are the mean gray value of different areas . Bresson transformed the original active contour model to a convex minimization problem by relaxing {0,1} to and showed that the characteristic function is the global minimizer . This model is based on mean gray value and it has drawbacks with different usages . For medical images we incorporate the edges and laplace information into active contour model for dealing with intensity inhomogeneities . The new model has the abilities of both (1) and (2). It is hard to segment medical images because the edges are always weak and the images are also noisy . We couple the higher - order diffusion model with active contour model for medical images segmentation . The new model that couples higher - order diffusion with active contour model is (3)arg min{\|\|dx dy+((1\|fu1\|2+1\|u1\|+1\|u1\|)+(2\|fu2\|2+2\|u2\|+2\|u2\|)(1))dx dy}, where is the image domain, 1, 1, 1, 2, 2, 2 are the positive parameters, f is the original image, u 1 and u 2 are the mean values in different image parts, and is a standard level set function . We use the form of (3) mainly because the image features in different areas are not always the same . This is a natural extension of chen and vese and vese and chen model for piecewise constant or smooth model . We can get the solution using alternating iteration . Because there are gradient and laplace information, straightforward solving of (3) by introducing forth - order term of difference scheme is a hard work . The split bregman method [17, 18] is introduced for the easy implementation of the model . We introduce the auxiliary variables w1=(w11,w12)t, w 2 and bregman iteration parameters b=(b1,b2)t, when the following energy function gets its minimization, w1u, w2u . Equation (3) can be divided into the following three sub - problems of minimization: fix and u 2, the energy function of (3) became the following form: (4)(u1k+1,w1k+1,w2)=arg minw1,u1{(1\|w1\|+1\|w2\|+1\|fu1\|2+12(w1u1b1k)2+12(w2w1)2) dx dy}, where w1k+1=u1k+1, b1k+1=b1k+u1k+1-w1k+1, w2=w1, 1, and 1 are the positive parameters . The energy function of u 1 is (5)e(u1)=(1\|fu1\|2+12(w1u1b1k)2) dx dy . The discretion of u 1 is (7)u1=1(21 + 41)(21f+1(u1,i, j+1k+u1,i, j1k + u1,i+1,jk+u1,i1,jk + (b1kw1k))). The energy function of w1 is (8)e(w1)=(1\|w1\|+12(w1u1b1k)2 + 12(w2w1)2) dx dy . Using eular - lagrange equation, we can get (9)w1k+1=uk+1+bk+1+11(w1w2)11w1k+1\|w1k+1\| . By wavelet soft threshold (10)w1k+1=max(\|u1k+1+b1k+11(w1w2)\|11,0)u1k+1+b1k+(1/1)(w1w2)\|u1k+1+b1k+(1/1)(w1w2)\| . The energy function of w 2 is (11)e(w2)=(1\|w2\|+12(w2w1)2) dx dy . Using eular - lagrange equation, we can get (12)w2k+1=w1k+111w2k+1\|w2k+1\| . By wavelet soft threshold, (13)w2k+1=max(\|w1k+1\|11,0)w2k+1\|w2k+1\| . For solving u 2 the energy function can be rewritten as: (15)arg min{e()=\|\|dx dy+r(u1,u2) dx dy}. For solving, we also use the split bregman method by introducing v=. Then the energy function of (14) is transformed as: (16)(k+1,vk+1) = arg mind,{\|v\|dx dy + r(u1,u2) dx dy + 2(vdk)2dx dy}, where dk+1=dk+k+1-vk+1, is the positive parameter . Fixing v for solving: (17)r(u1,u2)(k+1+dkvk)=0 . Using gradient descent method, (18)ddt=(k+1+dkvk)r(u1,u2). After discretion, (19)k+1 = (11 + 4dt)dt ((i, j1k+i, j+1k+i1,jk+i+1,jk+dkvk) r(u1,u2)). To ensure that, so in every step we use the following function: (20)k+1=min(max(k+1,0),1). Fix for solving v, we can get (21)vk+1=max(\|k+1+dk\|1,0)k+1+dk\|k+1+dk\| . In the end of calculation, we set (22)(x)={0(x) th 1(x)<th, where th is the preset threshold . To verify the effect of our proposed method, we test our method on a variety of real images with intensity inhomogeneity . We compare our method with cv model and mean shift algorithm because they are highly cited and compared in image segmentation . The cv model which is used in our experiment for comparison is also global convex . For comparison with the mean shift algorithm, we used the software edison which is based on a fast implementation of the mean shift algorithm using a speed - up scheme described in [1921]. Figure 1 shows the results for a real image of a t - shaped object with different lighting intensity . Figure 4 is a test of mr image of blades with the comparison of cv model and mean shift method . All of them are selected because they are typical images with intensity inhomogeneity . The cv model and mean shift method cannot get the proper results while our model can get the right results . The shadow of the object is the key element causing wrong segmentation of tradition methods . The vessel images in figures 2 and 3 fail to be segmented using traditional methods because of the intensity inhomogeneity . But using our model, the boundary of the object of interest (the bladder) is extracted very well . In these two images, parts of the vessel boundaries are quite weak; our method can still segment them well . The result of mean shift algorithm for the first image is similar to that of our method, showing certain ability of the mean shift algorithm in handling intensity inhomogeneity . However, for the two - vessel image, a small portion of the vessel is missing . For segmenting the mr bladder image, the result is not so perfect for surrounding organs . We notice that the segmentation result of mean shift algorithm is somewhat sensitive to the choice of two major parameters: spatial bandwidth and range bandwidth . We have tweaked these two parameters and other minor parameters for the best segmentation results for these four images . In this paper, we present a new active contour model for medical image segmentation . It can segment images with intensity inhomogeneity and has desirable performance for images with weak object boundaries . Experimental results have demonstrated the advantages of our method over several well - known methods for image segmentation.
The study was conducted in the stem cell facility, all india institute of medical sciences (aiims), new delhi, india, after obtaining prior approval of the study protocol by the institute ethics committee . Preparation of porous 3d scaffolds: chitosan, hydroxyapatite and polycaprolactone were supplied by sigma alderich, usa . The molecular weight and melt index of pcl were specified as 48,000 - 90,000 da and 1.8 (g/10 min, 80c). The molecular weight and density for cht were 70,000 - 90,000 da and 1.145 g / ml at 25c, respectively . The molecular weight and particle size of hap were 502.31 da and <200 nm, respectively . The pure chitosan, designated as 100cht, was selected as matrix material for the preparation of the composites . The composites, cht / hap / pcl in the ratio of 60/0/40, 60/10/30 and 60/15/25 designated as 40cht / hap, 30cht / hap / pcl and 25cht / hap / pcl, respectively, were prepared by freeze drying technique using a lyophilizer (coolsafe 110 - 4, labogene, lynge, denmark). The chitosan was dissolved in 2 mm acetic acid and pcl was dissolved in glacial acetic acid (fisher scientific, mumbai, india) at 45c using magnetic stirrer . The gel - like mixture was casted in a glass petri dish and freeze dried at -80c for 24 h. these samples were then neutralized in sodium hydroxide (naoh, hi - media, mumbai, india) solution and washed with deionized water to remove any remaining naoh . The scaffolds were cut into disc of 10 mm diameter and 2 mm thickness for in vitro studies . Morphology of scaffolds - the microstructure of the scaffolds was examined by stereo - scan 360 scanning electron microscope (sem, cambridge scientific industries, ma, usa). Diameter of individual pores in the scaffolds were measured directly from the sem images with 100x magnification using nih image j software (dr wayne rasband, national institutes of health, bethesda, porosity measurement of scaffolds - the porosity of scaffolds was measured by liquid displacement method10 . The procedure for liquid displacement method was done as follows: the volume (v0) and weight (w0) of the samples were measured . Then, the sample was immersed into absolute ethanol until it was saturated by it . The porosity of the scaffold was calculated according to the following equation: degree of porosity (%) = 100 x (w1-w0)/ v0 (represents the density of the ethanol). In vitro degradation of scaffolds - the ability of scaffolds to degrade in vitro was studied using a lysozyme (sigma alderich, usa) degradation test . Lysozyme was used for the degradation study in the concentration similar to the circulatory level in human body (to mimic in vivo condition). The samples were then placed in 0.1 m pbs (phosphate buffered saline) ph 7.4 containing 100 mg / l of lysozyme and incubated at 37c . The samples were removed at 1, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days to evaluate the weight loss . Per cent weight loss was calculated according to the following equation:% weight loss = (w0-wt) / w0x100 where, w0 is the starting dry weight and wt is the dry weight at specified time . Mechanical properties of scaffolds - compressive properties of the scaffolds in wet state were evaluated with a universal testing machine model h5ks (tinius olsen, surrey, england, uk) with qmat 5.37 professional software . The specimens were prepared as column of 20 mm in diameter and 10 mm in height . The scaffolds were immersed in pbs at ph 7.4 and 37c for three days for complete hydration . The crosshead speed of the machine was set at 1mm / min with a load cell of 1 kilonewton until 50 per cent reduction in specimen height . Culture and seeding of the cells on scaffolds - human bm was collected from the iliac crest of the patients undergoing stem cell transplantation after taking prior consent . Briefly, 1 - 2 ml of bm was mixed with expansion media in 1:1 ratio and plated on the culture flasks . After incubation at 37c for 24 h non - adherent cells were removed and fresh medium was added . In our experiments, hmscs were cultured and expanded in a non - differentiating growth medium consisting of low glucose dulbecco's modified eagle medium (dmem, gibco, life technologies, usa) supplemented with 10 per cent foetal bovine serum (fbs) (0.03 m, hyclone, thermo scientific, usa), 2 mm of l - glutamine (gibco, life technologies, usa), 100 units / ml of penicillin and 0.1mg / ml of streptomycin (gibco, life technologies, usa). Cells were grown in a 5 per cent co2 atmosphere at 37c, and the medium was renewed every three days . Before confluence, cells were detached using trypsin - edta (gibco, life technologies, usa) and passaged 1:3 into fresh culture flasks . These cells were characterized using bd - lsr ii (becton - dickinson, usa) and for their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages . The cells were positive for the cell surface markers cd105, cd29, (ebioscience, usa), cd73 and cd90 and negative for hla class ii and cd34/45 (bd pharmingen, usa). Hmscs were grown to 80 per cent confluency, trypsinized, and re - suspended in cell expansion medium . Hmscs (5x10/20 l) were seeded drop wise onto sterilized scaffold and incubated at 37 c for 3 h to allow the cells to adhere, then 2 ml of medium was added to each well . The seeded scaffolds were cultured for four weeks with medium change thrice a week . For osteogenic differentiation, the cell expansion medium was supplemented with 10 nm dexamethasone, 50 g / ml ascorbic acid- 2 phosphate and 10 mm beta - glycerophosphate obtained from sigma, usa . For cytotoxicity studies mouse lung fibroblast l929 cells were procured from national centre for cell science, pune, india . The cells were grown in dmem (gibco, life technologies, usa) supplemented with 10 per cent fbs and 100 units / ml of penicillin and 0.1mg / ml of streptomycin in a humidified incubator . Cytotoxicity evaluation of scaffolds - the cytotoxicity of the scaffolds was assessed by [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (mtt) assay . For cytotoxicity evaluation, we adopted the procedure from the iso10993 - 5 standard test method21 . Extraction media were prepared by immersing dry specimens in cell culture medium as described in literature21 . L929 and hmscs were separately cultured in 48 well (5x10/cells / well) plate in 500 l extraction medium . After 24 h, mtt (sigma, usa; 5 mg / ml in pbs) was added to each well and incubated for 3.5 h at 37c . Then, dimethylsulphoxide (sigma, usa) was added to each well to dissolve the formazan crystals produced by the activity of live cells and the coloured supernatant was read at 570 nm using plate reader (el 800, biotek, usa). In addition, standard calibration curve was made by mtt assay on known number of cells and the absorbance values were plotted against the known cell numbers . Metabolically active cell numbers were then determined using this standard curve based on their mtt absorbance . Cell viability and distribution over scaffold - viability and distribution of the cells seeded in the scaffold were examined after 7 and 14 days using live / dead staining kit (k501 - 100, biovision, ca, usa). Scaffolds seeded with hmscs (5x10 cells / scaffold) were washed with pbs and incubated with fluorescene di acetate (fda) and propidium iodide (pi). After staining, the cells were visualized using confocal laser scanning microscope (clsm) (tcs sp5, leica, germany). Cell attachment and morphology - cell attachment and morphology of cells seeded on the scaffolds were examined by scanning electron microscopy (sem). Samples were collected at different time points and fixed with 2.5 per cent glutaraldehyde and stored at -80 c . Were mounted over aluminium stubs and sputter - coated with gold prior to imaging with a leo 435 vp scanning electron microscope (co - operation zeiss, leica, cambridge, uk) at 5 kva in secondary electron imaging mode . Alkaline phosphatase (alp) activity - the differentiation of hmscs into osteoblasts in the scaffolds was evaluated by the measurement of alkaline phosphatase activity22 . Briefly, the samples were harvested and freeze - thawed thrice in lysis buffer (1.0% triton x-100) for cell lysis . An aliquot of the lysate was added to the alp substrate buffer (equal parts of 20 mm p - nitrophenyl phosphate (1.5 m 2-amino-2-methyl-1-propanol from sigma, usa, and 10 mm mgcl2), and incubated for 30 min at 37 c . Then hydrolysis of the substrate to p - nitrophenol was measured spectrophotometrically at 405 nm (el 800, biotek, usa). Quantitative reverse transcription - pcr (qrt - pcr) analysis for the relative expression of osteoblasts specific genes, in differentiated osteoblasts derieved from hmscs - the expression levels of collagen type 1 (colia1), alkaline phosphatase (alp), osteocalcin (oc), osteopontin (op), briefly, at each time point, total rna was isolated using trizol (life technologies, usa) as per the manufacturer's protocol . Rna samples were treated with dnase i (sigma; 1 u/g rna) for 25 min at room temperature to remove residual genomic dna . The concentration of the purified rna was quantified using nanospectrophotometer (p300, implen gmbh, germany). Cdna was transcribed using 200u of m - mlv reverse transcriptase, 0.5 mm dntps and 12.5 ng / ml oligo - dt and 40 u of rnase inhibitor rnasin (all from promega, usa). The gene specific primers were custom made (sigma, bangalore, india) and are summarized in table i. reactions were carried out using sybr green super mix (kapa biosystems, capetown, south africa) in a final volume of 10 l with 0.3 m of each primer . Relative gene expression was quantified by 2 method24 in which the accumulated pcr products of each of the genes examined was normalized to the housekeeping gene gapdh in the corresponding samples . Ct was obtained by subtracting the ctgapdh from cttarget (ct = cttarget ctgapdh) and ct was obtained by subtracting ctuninduced cells from ctinduced cells (ct = ctinduced cells- ctun induced cells). Negative controls were used as no template cdna reactions and melting curves were used to confirm the results . Primer sequence used for qpcr amplification the relative quantitative qpcr experiments were performed by using a realplex 4 epgradient mastercycler (eppendorf, hamburg, germany) according to the manufacturer's instructions . Statistical analysis: all statistical analyses were performed using graphpad software (la jolla, ca, usa). All measurements were collected in triplicates and expressed as means standard deviation, unless otherwise noted . In cases where statistical comparisons were made between three or more groups of data, a one - way analysis of variance morphology of scaffolds - the microstructure of the scaffolds was examined by stereo - scan 360 scanning electron microscope (sem, cambridge scientific industries, ma, usa). Diameter of individual pores in the scaffolds were measured directly from the sem images with 100x magnification using nih image j software (dr wayne rasband, national institutes of health, bethesda, porosity measurement of scaffolds - the porosity of scaffolds was measured by liquid displacement method10 . The procedure for liquid displacement method was done as follows: the volume (v0) and weight (w0) of the samples were measured . Then, the sample was immersed into absolute ethanol until it was saturated by it . The porosity of the scaffold was calculated according to the following equation: degree of porosity (%) = 100 x (w1-w0)/ v0 (represents the density of the ethanol). In vitro degradation of scaffolds - the ability of scaffolds to degrade in vitro was studied using a lysozyme (sigma alderich, usa) degradation test . Lysozyme was used for the degradation study in the concentration similar to the circulatory level in human body (to mimic in vivo condition). The samples were then placed in 0.1 m pbs (phosphate buffered saline) ph 7.4 containing 100 mg / l of lysozyme and incubated at 37c . The samples were removed at 1, 3, 7, 14, 21, 28, 35, 42, 49 and 56 days to evaluate the weight loss . Per cent weight loss was calculated according to the following equation:% weight loss = (w0-wt) / w0x100 where, w0 is the starting dry weight and wt is the dry weight at specified time . Mechanical properties of scaffolds - compressive properties of the scaffolds in wet state were evaluated with a universal testing machine model h5ks (tinius olsen, surrey, england, uk) with qmat 5.37 professional software . The specimens were prepared as column of 20 mm in diameter and 10 mm in height . The scaffolds were immersed in pbs at ph 7.4 and 37c for three days for complete hydration . The crosshead speed of the machine was set at 1mm / min with a load cell of 1 kilonewton until 50 per cent reduction in specimen height . Culture and seeding of the cells on scaffolds - human bm was collected from the iliac crest of the patients undergoing stem cell transplantation after taking prior consent . Briefly, 1 - 2 ml of bm was mixed with expansion media in 1:1 ratio and plated on the culture flasks . After incubation at 37c for 24 h non - adherent cells were removed and fresh medium was added . In our experiments, hmscs were cultured and expanded in a non - differentiating growth medium consisting of low glucose dulbecco's modified eagle medium (dmem, gibco, life technologies, usa) supplemented with 10 per cent foetal bovine serum (fbs) (0.03 m, hyclone, thermo scientific, usa), 2 mm of l - glutamine (gibco, life technologies, usa), 100 units / ml of penicillin and 0.1mg / ml of streptomycin (gibco, life technologies, usa). Cells were grown in a 5 per cent co2 atmosphere at 37c, and the medium was renewed every three days . Before confluence, cells were detached using trypsin - edta (gibco, life technologies, usa) and passaged 1:3 into fresh culture flasks . These cells were characterized using bd - lsr ii (becton - dickinson, usa) and for their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages . The cells were positive for the cell surface markers cd105, cd29, (ebioscience, usa), cd73 and cd90 and negative for hla class ii and cd34/45 (bd pharmingen, usa). Hmscs were grown to 80 per cent confluency, trypsinized, and re - suspended in cell expansion medium . Hmscs (5x10/20 l) were seeded drop wise onto sterilized scaffold and incubated at 37 c for 3 h to allow the cells to adhere, then 2 ml of medium was added to each well . The seeded scaffolds were cultured for four weeks with medium change thrice a week . For osteogenic differentiation, the cell expansion medium was supplemented with 10 nm dexamethasone, 50 g / ml ascorbic acid- 2 phosphate and 10 mm beta - glycerophosphate obtained from sigma, usa . For cytotoxicity studies mouse lung fibroblast l929 cells were procured from national centre for cell science, pune, india . The cells were grown in dmem (gibco, life technologies, usa) supplemented with 10 per cent fbs and 100 units / ml of penicillin and 0.1mg / ml of streptomycin in a humidified incubator . Cytotoxicity evaluation of scaffolds - the cytotoxicity of the scaffolds was assessed by [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (mtt) assay . For cytotoxicity evaluation extraction media were prepared by immersing dry specimens in cell culture medium as described in literature21 . L929 and hmscs were separately cultured in 48 well (5x10/cells / well) plate in 500 l extraction medium . After 24 h, mtt (sigma, usa; 5 mg / ml in pbs) was added to each well and incubated for 3.5 h at 37c . Then, dimethylsulphoxide (sigma, usa) was added to each well to dissolve the formazan crystals produced by the activity of live cells and the coloured supernatant was read at 570 nm using plate reader (el 800, biotek, usa). In addition, standard calibration curve was made by mtt assay on known number of cells and the absorbance values were plotted against the known cell numbers . Metabolically active cell numbers were then determined using this standard curve based on their mtt absorbance . Cell viability and distribution over scaffold - viability and distribution of the cells seeded in the scaffold were examined after 7 and 14 days using live / dead staining kit (k501 - 100, biovision, ca, usa). Scaffolds seeded with hmscs (5x10 cells / scaffold) were washed with pbs and incubated with fluorescene di acetate (fda) and propidium iodide (pi). After staining, the cells were visualized using confocal laser scanning microscope (clsm) (tcs sp5, leica, germany). Cell attachment and morphology - cell attachment and morphology of cells seeded on the scaffolds were examined by scanning electron microscopy (sem). Samples were collected at different time points and fixed with 2.5 per cent glutaraldehyde and stored at -80 c . Were mounted over aluminium stubs and sputter - coated with gold prior to imaging with a leo 435 vp scanning electron microscope (co - operation zeiss, leica, cambridge, uk) at 5 kva in secondary electron imaging mode . Alkaline phosphatase (alp) activity - the differentiation of hmscs into osteoblasts in the scaffolds was evaluated by the measurement of alkaline phosphatase activity22 . Briefly, the samples were harvested and freeze - thawed thrice in lysis buffer (1.0% triton x-100) for cell lysis . An aliquot of the lysate was added to the alp substrate buffer (equal parts of 20 mm p - nitrophenyl phosphate (1.5 m 2-amino-2-methyl-1-propanol from sigma, usa, and 10 mm mgcl2), and incubated for 30 min at 37 c . Then hydrolysis of the substrate to p - nitrophenol was measured spectrophotometrically at 405 nm (el 800, biotek, usa). Quantitative reverse transcription - pcr (qrt - pcr) analysis for the relative expression of osteoblasts specific genes, in differentiated osteoblasts derieved from hmscs - the expression levels of collagen type 1 (colia1), alkaline phosphatase (alp), osteocalcin (oc), osteopontin (op), transcription factor cbfa 1 and osterix genes were quantitated23 . Briefly, at each time point, total rna was isolated using trizol (life technologies, usa) as per the manufacturer's protocol . Rna samples were treated with dnase i (sigma; 1 u/g rna) for 25 min at room temperature to remove residual genomic dna . The concentration of the purified rna was quantified using nanospectrophotometer (p300, implen gmbh, germany). Cdna was transcribed using 200u of m - mlv reverse transcriptase, 0.5 mm dntps and 12.5 ng / ml oligo - dt and 40 u of rnase inhibitor rnasin (all from promega, usa). The gene specific primers were custom made (sigma, bangalore, india) and are summarized in table i. reactions were carried out using sybr green super mix (kapa biosystems, capetown, south africa) in a final volume of 10 l with 0.3 m of each primer . Relative gene expression was quantified by 2 method24 in which the accumulated pcr products of each of the genes examined was normalized to the housekeeping gene gapdh in the corresponding samples . Ct was obtained by subtracting the ctgapdh from cttarget (ct = cttarget ctgapdh) and ct was obtained by subtracting ctuninduced cells from ctinduced cells (ct = ctinduced cells- ctun induced cells). Negative controls were used as no template cdna reactions and melting curves were used to confirm the results . Primer sequence used for qpcr amplification the relative quantitative qpcr experiments were performed by using a realplex 4 epgradient mastercycler (eppendorf, hamburg, germany) according to the manufacturer's instructions . Statistical analysis: all statistical analyses were performed using graphpad software (la jolla, ca, usa). All measurements were collected in triplicates and expressed as means standard deviation, unless otherwise noted . In cases where statistical comparisons were made between three or more groups of data, a one - way analysis of variance was performed . In cases where only two sets of data were compared, microstructure of scaffolds: the external morphology of scaffolds was studied by sem (fig . Scaffolds were seen to be highly porous with open and interconnected pores surrounded by thin polymer walls . The microstructure of the scaffolds did not seem to vary from one another since the treatment given to each was similar . The composition did not affect the pore size and porosity of the scaffolds . Pore size and porosity data of the scaffolds are given in table ii . Representative scanning electron microscope (sem) photomicrographs illustrating the open porous structure of the scaffolds with homogenous distribution of pores . (a) 100cht (b) 40cht / hap (c) 30cht / hap / pcl and (d) 25cht / hap / pcl (scale bar 100m). Pore parameters of scaffolds, determined by scanning electron microscopy and liquid displacement methods in vitro degradation of scaffolds: fig . 2a shows the weight loss of scaffolds as a function of soaking time in pbs . The 100cht showed maximum weight loss (91%), followed by 40cht / hap (70%), 30cht / hap / pcl (34%) and 25cht / hap / pcl (30%) at the end of 56 days . After enzymatic degradation for 56 days, change in morphology of the 100cht and 40cht / hap scaffolds was observed (fig . The scaffold 30cht / hap / pcl and 25cht / hap / pcl remained in the original shape, indicating that these scaffolds had good structural stability . (a) degradation kinetics of 100cht, 40cht / hap, 30cht / hap / pcl and 25cht / hap / pcl scaffolds . Data represent mean value of five independent experiments, (b) digital images showing the morphology of scaffolds after in vitro enzymatic treatment at days 1 and 56 . Mechanical properties of scaffolds: the compressive stress - strain curves of the pure and composite scaffolds are shown in fig . 3, and the data of compressive strength and compressive modulus are given in table iii . As expected, 100cht scaffold showed low compressive strength and modulus . The mixing of hap with cht significantly improved the compressive strength and compressive modulus of 40cht / hap . Further addition of pcl (10%) to the 40cht / hap showed no significant difference in the compressive strength and modulus of the 30cht / hap / pcl composite scaffold . Increasing the pcl content to 15 per cent dramatically increased the compressive strength (p<0.01) and compressive modulus (p<0.001) of 25cht / hap / pcl composite scaffold . Mechanical properties of porous scaffolds cytotoxicity of the scaffolds: the cytotoxicity of 3d composite scaffolds was tested by quantitative analysis using the mtt assay . The absorbance values of test samples after exposure to the extraction medium for 24 h were similar to the negative control (table iv). Absorbance values correspond to the number of metabolically active cells . Thus our results (fig . 4) indicated that the metabolic activity of l929 and hmscs cells was not inhibited by the extraction medium . From the quantitative scores, it was concluded that the extracts of the scaffolds demonstrated no cytotoxic reactivity in this test . The viability of mouse lung fibroblast cell line l929 and hmscs was evaluated that had been cultured for 24 h with the extraction media from the specimens . Viability and distribution of hmscs in scaffolds: the viability and distribution of hmscs on scaffolds were visualized using clsm after 7 and 14 days of cells seeding, which depicted a high ratio of living cells on all scaffold compositions . As shown in fig . 5,> 90 per cent cells were live, randomly distributed within the porous 3d scaffolds . Hmscs were connected to each other and formed a cell network across the surface of the scaffolds . The cells proliferated and their number increased from day 7 to day 14 (fig . Up to 14 days of cultivation, no large difference in the viability and morphology was observed . Cells were more evenly distributed over 30cht / hap / pcl and 25cht / hap / pcl scaffolds compared to 100cht and 40cht / hap scaffolds . Confocal laser microscopic images of hmscs grown on scaffolds after seven days (a) cells were randomly distributed and forming patch on 100cht, (b) hmscs adhered and attained fibroblastic morphology on 40cht / hap, (c, d) cells were more evenly distributed over 30cht / hap / pcl and 25cht / hap / pcl, covered whole surface of the scaffold . Viability and distribution of cells were detected using fluorescence markers that allowed life / death stain (fda / pi): dead cells were stained red whereas viable cells were stained green (magnification 10x). (a) cells proliferated and form a large patch on 100cht, (b) cells increased in number and evenly distributed on 40cht / hap, (c, d) cells proliferated and their number increased from day 7 to 14 . Cells were connected to each other and formed a cell network across the surface of 30cht / hap / pcl and 25cht / hap / pcl scaffolds . Viability and distribution of cells were detected using fluorescence markers that allowed life / death stain (fda / pi): dead cells were stained red whereas viable cells were stained green . The qualitative image analysis revealed that a larger number of cells stayed viable throughout the entire culture period . Attachment and morphology of hmscs on scaffolds: after seven days of cell seeding, cells adhered and spreaded over the porous scaffold surface and merged completely with each other so that intercellular connections were not visible (fig . After 14 days it was found that cells were merged and formed a complete cellular layer on the surface of the scaffolds, so that the surface was almost covered and only a few pores were visible and a few cells migrated inside the pores (fig . Scanning electron microscope (sem) images of human mesenchymal stem cells (hmscs) grown on 100cht, 40cht / hap, 30cht / hap / pcl and 25cht / hap / pcl scaffolds after seven days . Sem images of hmscs grown on 100cht, 40cht / hap, 30cht / hap / pcl and 25cht / hap / pcl scaffolds after 14 days . At 14 days, cells grew and merged to form a cell sheet, got enlarged and migrated into the pores of scaffolds . (scale bar 30 m). Specific alkaline phosphatase activity of hmscs differentiated into osteoblasts on scaffolds: alp is an enzyme produced by differentiating osteoblasts . Alp activity can serve as a marker indicating differentiation of hmscs toward the osteoblastic lineage . In un - induced culture condition (fig . 9a), the alp activity of hmscs on scaffolds and tissue culture plastic surface (tcps) slightly increased over the whole culture period of 28 days . 9b) the alp activity of the hmscs, increased progressively over time from day 3 to 28 . Alp activity of hmscs in osteogenic condition was significantly higher on all scaffolds compared to tcps (p<0.01). Among the scaffolds, determination of alkaline phosphatase (alp) activity of hmscs over 100cht, 40cht / hap, 30cht / hap / pcl, 25cht / hap / pcl scaffolds and tissue culture plastic surface (tcps) under (a) un - induced culture condition and (b) induced culture condition . Alp activity of hmscs in the cell - scaffold constructs was assessed during four weeks of culture . Qpcr analysis for relative expression of osteoblasts specific genes, in osteoblasts derived from differentiation of hmscs: results obtained from qrt - pcr were quantitative and presented as a fold change in mrna levels in differentiated hmscs when compared to undifferentiated hmscs (fig . 10). Differentiation of osteogenic - induced hmsc on scaffolds and tcps was further demonstrated by an increase in the relative expression of the osteoblasts specific genes . The relative expression of osteoblasts specific genes on scaffolds was several fold upregulated compared to tcps . A slightly higher maximal expression of genes was detected on scaffold 25cht / hap / pcl compared to 30cht / hap / pcl, 40cht / hap and 100cht scaffolds (found on day 28). Real - time quantitative reverse transcriptase pcr analysis of the expression levels of osteogenic markers . Expression levels of cbfa-1, osterix, alkaline phosphatase, collagen type i, osteocalcin, and osteopontin genes were determined in hmscs cultured over 100cht, 40cht / hap, 30cht / hap / pcl and 25cht / hap / pcl scaffolds and tissue culture plastic surface (tcps) under osteogenic and non - osteogenic culture conditions for 28 days . In this study we prepared porous foam like scaffolds with average pore size of 140 m, which could support cell penetration, migration, tissue in - growth and vascularisation as reported in the literature2526 and the average porosity of the scaffold was 82 per cent, which was in the range of the porosity of the trabecular bone (50 - 90%). Mean pore sizes increase, mechanical strength is decreased . In this study, by fabricating scaffolds with optimal pore size and porosity it was possible to maintain the mechanical strength of scaffold . The porous 100cht scaffolds were tissue biocompatible but mechanically weak and unable to maintain structure integrity as also reported in literature11 . Absorbs a lot of water and swells resulting in poor structural stability and low mechanical strength . To overcome this problem we doped cht with pcl and hap . Pcl being hydrophobic decreased the total water absorption and swelling and hap as a filler strengthened the scaffold . The resulting composite 25cht / hap / pcl scaffold showed better compressive strength compared to pure 100cht scaffold, which was very close to the lower limit of compressive strength of trabecular bone (2 - 10 mpa)6 . Thus, incorporation of hap and pcl into composite scaffolds in specific ratios could improve their mechanical properties . In in vitro degradation study, it was found that the weight loss of the scaffolds increased gradually . The degradation rate of 100cht and 40cht / hap scaffold was high compared to the composite scaffolds, 30cht / hap / pcl and 25cht / hap / pcl . The result suggested that presence of pcl decreased degradation of the composite scaffold because of its being hydrophobic and semi - crystalline material with a slow in vitro hydrolytic degradation rate27 . Pure 100cht scaffolds were found to degrade> 90 per cent and disintegrated upon handling . In contrast, the composite 30cht / hap / pcl and 25cht / hap / pcl scaffolds retained their original shape, despite evidence of degradation from weight loss measurements . The mtt assay showed that all the scaffolds prepared in this study were nontoxic and biocompatible . The polymer composition and the method of fabrication also play an important role in cell biocompatibility . Our results showed overall good viability of the cells indicating that these scaffolds were free of toxic chemicals and suitable for in vitro and in vivo studies . The ability of hmscs to differentiate into osteogenic lineage is the key to the use of these cells to improve bone formation . Sem images showed that the large surface area of the porous scaffolds allowed hmscs to adhere, spread and grow on the scaffolds . It has been shown that chitosan coating on 2-d polymeric films or 3-d polymer scaffolds may affect the attachment, proliferation and differentiation of cells28 . The flat morphology, and excellent spreading in and around the interconnected porous structure, indicated strong cellular adhesion and growth of cells . The 3d structure of the scaffold might have been responsible for the attachment of hmscs and differentiation into osteoblasts29 . The parallel investigations of cell viability and distribution carried out by clsm revealed proliferation of the cells on the scaffolds with clear spreading and good morphology . Live dead staining and clsm showed that> 90 per cent hmscs were live and distributed throughout the scaffolds . This indicates that the pores of the scaffolds are interconnected and large enough for cells to penetrate and colonize the porous structure . Alp is an enzyme expressed by mscs during osteogenesis and is a well - defined marker of osteoblastic lineage30 . Thein - han et al16 has demonstrated that presence of hap nanoparticles in cht / nhap nanocomposite scaffolds influence cytoskeleton organization, which regulates cellular signal transduction, cell differentiation and gene expression . In this study, we also found that hmscs cultured over composite scaffolds showed higher specific alp activity than those cultured over pure cht scaffolds and tcps . The expression of osteoblasts specific genes was multiple folds in induced hmscs compared to the un - induced hmscs . However, in non - osteogenic conditions the cells over 3d scaffolds showed enhanced osteoblastic genes expression compared to cells cultured on tcps . It might be because of the inherent osteoinductivity and osteoconductivity of the cht and hap present in the scaffold . In addition, results of a cell culture study showed both enhanced proliferation and differentiation of rat calvarial osteoblasts on bone cement scaffold32 . After comparing all the properties of scaffolds it was found that the novel 25cht / hap / pcl biomedical composite scaffold had good mechanical strength, optimum pore size and porosity, required degradability, cell biocompatibility and osteo - inductivity . In conclusion, the present study demonstrated the feasibility of using the freeze drying technique to fabricate 3d biocomposite scaffolds . The addition of hap and pcl to cht improved the compressive strength while maintaining high porosity with interconnected pores . The in vitro biocompatibility study of the scaffolds showed that the polycaprolactone did not affect attachment, proliferation and differentiation on the scaffolds . The 25cht / hap / pcl composite scaffold possessed the inherent physiochemical and efficient mechanical and biocompatible characteristics . The 25cht / hap / pcl biocomposite appears to be potential scaffolds as an off the shelf, synthetic, resorbable bone graft substitute for bone regeneration.
In 2012, an estimated 790,000 women in the united states were diagnosed with cancer, of whom 10% were of reproductive age (american cancer society, 2012, schover, 2005). [nih surveillance] (national institute of health, 2010, society of family planning, 2012) addressing the possibility of future pregnancy is an important aspect of survivorship and quality of life for patients undergoing cancer treatment, and several ways to preserve fertility during cancer therapy have been developed and studied (letourneau et al ., 2012, as background, 49% of pregnancies in the united states are unintended and unplanned (the alan guttmacher institute (agi), 2013). Patients with medical co - morbidities, including those undergoing cancer treatment and/or surveillance, are among this group . It is estimated the incidence of malignancy and pregnancy is approximately 1:1000 (pavlidis, 2002, smith et al ., 2003). Although rare, pregnancy in the setting of cancer treatment may create clinical and ethical dilemmas, with potential increased risks for both the patient and gestation . These dilemmas are further compounded when a pregnancy in this setting is unplanned . Among women who have undergone or are currently undergoing cancer treatment, there is limited data regarding the rate of unintended pregnancy or patients' impression of fertility . It has been previously reported that childhood cancer survivors between 15 and 30 years old were more likely to terminate a pregnancy compared to age - matched controls (green et al ., 2002). A survey of female cancer patients revealed that 55% believed that they could not become pregnant after cancer treatment, and 45% of the same group denied using any contraception method (patel et al ., 2009). Both of the aforementioned studies support the notion that we could improve counseling in this patient population regarding fertility potential and contraception planning . In gynecologic cancer patients, women with early stage cancers may be eligible for fertility sparing treatment, and therefore may be at risk for unplanned pregnancy after therapy if contraception is not addressed as well (mclaren and bates, 2012). For this reason, we conducted a survey of current members of the society of gynecologic oncology (sgo) regarding reproductive counseling, practices, and experience with unplanned pregnancy in this population . After obtaining approval from our institutional review board, a list of member email addresses was obtained from the sgo . An email statement of confidentiality with the questionnaire link was emailed to all listed members . Following the initial e - mail, members received two additional follow - up e - mails, two and four weeks respectively, after the initial contact . The survey remained open for responses for two weeks following the third, and final, reminder . Survey monkey was utilized to create and administer a nineteen question survey regarding demographics, contraception counseling and use practices, referral patterns, and incidence of unplanned pregnancy . Demographic data queried were age, gender, region of country of current residence, medical specialty, level of training, description of clinical practice, and number of years caring for gynecologic oncology patients . Questions regarding consistency to which the provider addresses contraception and fertility concerns were answered using a likert - type scale (i.e., always, sometimes, rare, never, not applicable). The remaining questions were answered using a yes / no or a multiple - choice format . The multiple - choice questions specifically addressed the frequency of contraception methods counseling and administration, and number of patients experiencing unplanned pregnancy . Participants were asked to check all contraceptive methods that they counseled and administered / prescribed . Contraceptive methods specifically included were the oral contraception pill (ocp), injectable, intrauterine device (iud), or subdermal levonorgestrel implant . Prevalence of unplanned pregnancy was evaluated by asking practitioners to estimate prevalence of unplanned pregnancy among their patients (during or after treatment) in the previous one and five years respectively . We excluded the responses of anyone who reported that he / she was retired or did not provide information on their reproductive counseling patterns . Logistic regression analysis was used to model separately the odds of always or sometimes offering fertility and/or contraceptive counseling . All analyses were conducted using stata 13.0 (statacorp, college station, texas). Questionnaires were completed by 261 respondents, yielding a response rate of 18% . Among the respondents 21 (8%) reported that they were retired and/or did not provide information regarding their reproductive counseling patterns and thus were excluded from the analysis . Overall, the respondents had a mean age of 47 years with a slight predominance of females (52%), which is consistent with sgo demographics (mean age of male members 50 years old, mean age of female members 45 years old). Our survey had a higher proportion of fellow respondents (24%) than sgo as a whole (9%) [gynecologic oncology 2015: state of the subspecialty] selected characteristics of respondents are presented by reproductive counseling status in table 2 . Among respondents, 34.6% reported sometimes or always providing counseling on fertility and contraception, 19.2% reported providing fertility counseling only, 15.8% reported providing contraception counseling only, while 30.4% reported not routinely providing counseling on either topic . In general, providers who reported providing reproductive counseling on neither topic were more likely to be male and/or a fellow . They also tended to be <40 years of age, though this difference was not statistically significant . Physicians who reported not routinely counseling on either topic were less likely to report prescribing or administering contraception and/or were less likely to report referring to reproductive endocrinology . In contrast, those who reported providing counseling on both fertility and contraception were more likely to be female and/or an attending . They also were more likely to be older; although that difference was not statistically significant . Providers who reported sometimes or always providing counseling on both reproductive topics were also more likely to report providing or administering contraception and/or to refer to reproductive endocrinology . There were no notable differences in reproductive counseling status by u.s . Next, we examined what factors were associated with an increased odds of reporting providing fertility counseling sometimes or always (table 3). After adjustment for gender, we found that the odds of reporting providing fertility counseling were nearly three times higher among attending physicians as compared to fellows [aor = 2.72; 95% ci = (1.44, 5.12)]. In examining contraceptive counseling (table 4), we found that the odds of reporting providing contraceptive counseling was 2.8 times higher in women as compared to men and was 4.91 times higher in individuals age 50 + compared to those <40 after adjustment for level of practice . Overall, 81.7% of providers reported counseling, prescribing, or administering contraception to their patients . Among these individuals, the most frequently cited contraceptive that providers reported addressing with their patients included: oral contraceptive pills (81%), intrauterine devices (73%), depot medroxyprogesterone acetate (dpma) injections (56%), and contraceptive implants (21%). The most frequently cited contraceptives that providers reported prescribing or administering included: oral contraceptive pills (80%), intrauterine devices (62%), dpma injections (48%), and contraceptive implants (9%). The reported prevalence of unplanned pregnancy among the surveyed providers' patients was relatively rare, perhaps because it is not routinely addressed . Most providers (95%) reported 05 unplanned pregnancies among their patients in the last 5 years, with only 3 providers (1%) reporting more than ten unplanned pregnancies . Unplanned pregnancy in the setting of cancer treatment or surveillance is a complicated issue, and may create clinical and ethical dilemmas for the patient, partner, family, and treatment team . The majority of survey respondents appropriately acknowledged the risk of unplanned pregnancy among gynecologic cancer patients maintaining fertility potential . However, only half reported addressing contraception planning in this population on a consistent basis (52% always addressing contraception, versus 35% sometimes). Regarding the consistency with which contraception is addressed, these findings are consistent with a retrospective chart review completed at our institution where 45% of initial consultations documented a contraception plan in fertile patients of reproductive age, and 32% of follow - up visits for those that maintained fertility potential after cancer treatment (crafton et al ., 2016). Depending on individual circumstances patient's reproductive goals should be determined in order to tailor fertility - sparing treatment, when possible, versus contraception planning . Unfortunately, only half of respondents reported always addressing fertility concerns with patients in this population . This is consistent with the fact 59% of those surveyed routinely offer referral for preconception counseling for those patients considering fertility preservation options or planned pregnancy . Referral specifically for contraception counseling was reported less frequently, and even fewer reported follow up for plan establishment . Our study was unable to determine which patients or providers relied on the patient's primary care physician or gynecologist for the establishment of a contraception plan . The incidence of unplanned pregnancy in this population is reportedly rare, with 96% of responding physicians experiencing <5 unplanned pregnancies in the previous five years . This may be under - reported, as oncologists may be unaware of pregnancies, especially during disease surveillance . Despite the rarity, eleven providers report experiencing an estimated 620 unplanned pregnancies during that time same . The opportunity to avoid even a fraction of those merits further acknowledgement of this topic . It is reassuring that the large majority of providers reportedly counsel for and administer contraception methods, including 89% reportedly counseling for the iud and 77% employing its use . However, it was not specified in this survey if the indication for oral contraceptive pills (ocps) or an iud was for contraception planning or cancer therapeutic purposes . Of reported methods, ocps were the most frequent contraception method both counseled for and administered . When compared to implantable methods, both the iud and subdermal implant, both actual and ideal use of ocps have a higher failure rate, and therefore more reliable methods should be considered first, barring contraindications (centers for disease control and prevention, 2010). Given our findings that the attending cohort was more likely to provide contraception and/or fertility counseling sometimes or always compared to the fellow respondents (table 2, table 3), there seems to be an opportunity to improve education for fellows regarding fertility preservation options for these patients . As seen in other survey - based studies, the primary limitation of the study is the modest response rate, despite multiple recruitment attempts and limiting the length of the survey (cunningham et al ., 2015). Given the response rate, the potential for selection and survey content bias (i.e., only those who were interested in the topic responded) is present thus the results may have limited external validity . However, as aforementioned, our respondent cohort is comparable to the data published in the 2015 society of gynecologic oncology: state of the subspecialty . Inclusion of both fellows in training and retired members also limits the external validity of the survey regarding current members, but we felt inclusions of these populations were interesting and important in order to compare potential generational differences . Many gynecologic cancer diagnoses are made in peri - menopausal or postmenopausal women; therefore assessing what portion of a provider's practice is of reproductive age could aid discussion and interpretation of results . Likewise, as the type of cancer and respective treatment options may change the potential for or etiology of infertility, assessing providers' experience with specific disease processes, such as gestational trophoblastic neoplasia, also could aid discussion, and intervention . We acknowledge that a contraception plan may not appropriate for all patients undergoing cancer care, especially for who decline fertility sparing treatment, have been previously sterilized, or are actively attempting conception / currently pregnant . However, routine recognition of reproductive goals should be addressed by providers to alleviate potential biases or assumptions regarding patients' reproductive goals or sexual activity . Previous literature has reported providers' lack of counseling and patients' misunderstanding of reproductive potential after cancer treatment, and therefore a patient survey would be as valuable as the provider's impression we report (patel et al ., 2009, karaoz et al ., 2010). As life expectancy following cancer treatment diagnosis and treatment improves, and quality of life of survivors is emphasized, helping women meet their reproductive goals should remain an important focus . Comprehensive reproductive counseling should be emphasized, including both fertility sparing options and contraception planning, with the ultimate goal of decreasing unplanned pregnancy.
Thyroid cancer is the most common type of cancer in korea, with differentiated thyroid cancer (dtc, includes papillary and follicular thyroid cancer) accounting for the vast majority of cases . The standard treatment for dtc is thyroidectomy followed by i (radioactive iodine, rai) therapy and thyroxine suppression to decrease serum thyroid stimulating hormone (tsh). Rai therapy eliminates microscopic residual tissues after thyroidectomy which decreases the likelihood of thyroid cancer recurrence . Prior to rai therapy, the patient is advised to maintain a low - iodine diet (lid) to facilitate i uptake, maximizing the efficiency of rai therapy . Appropriate levels of dietary iodine intake or duration of the lid are not yet standardized over regions or countries . The american thyroid association recommends the lid with less than 50 g / day of dietary iodine for 1 - 2 weeks prior to rai therapy, but the korean thyroid association recommends a low - iodine diet with less than 100 g / day and provide a dietary guideline for the lid where allowed or restricted food items are listed during the lid . In iodine - rich areas such as korea, the recommended level of iodine intake during the lid is important to facilitate maintaining the lid, because a strict guideline may negatively impact the lid compliance . A few studies were reported results of less strict diet or short periods of the lid, however, the general consensus among clinical dietitians in korea regarding appropriate degree or duration of the lid have not yet elucidated . According to moon et al ., clinical dietitians should develop a practical dietary strategy in order to improve and facilitate the lid compliance . To improve the compliance of the lid most previous studies used urinary iodine measurements due to the lack of an iodine database and the difficulties in dietary survey . Urinary iodine excretion is a good measure for dietary iodine intake, but evaluating the dietary iodine intake using multiple days of dietary records would provide a more comprehensive measure of dietary iodine intake in order to develop a practical dietary strategy for the lid . Therefore, this study aims to assess dietary iodine level using three - day of dietary records and to search food sources that cause low adherence to the lid . In addition, we attempted to evaluate the ablation rate of remnant thyroid by compliance of the lid . We experienced three patients showing the diet composing iodine levels over 100 g / day despite their efforts for the lid before initial rai therapy . The protocol of case report was approved by the institutional review board of college of medicine at seoul national university (h-1308 - 066 - 513), and all the patients provided their written informed consent to participate . The patients had stage ii or iii papillary thyroid cancer, classified based on the cancer staging manual in the 7 edition of the american joint committee on cancer (ajcc). All three patients were educated on the preparation and management of the lid prior to rai therapy in a two - and - a - half - hour intensive education session . During the session, a clinical dietitian explained patients of the importance and methodology of maintaining the lid in a half - hour . Then the patients maintained the lid for two weeks prior to i administration (1.1 gbq) and a whole body scan (wbs), which was done three days after the i administration . To elevate their tsh levels, recombinant human tsh (rhtsh; thyrogen, genzyme, cambridge, ma, usa) dietary intakes for patients were assessed using three - day dietary records during usual diet period and the lid period respectively . Patients were taught how to record their diet and asked to record three - day dietary records at two weekdays and one weekend day . Energy and nutrient intakes, except for iodine, were calculated using a diet evaluation system (des). Iodine intake was estimated using the database recently established by han et al . And modified for this study . Successful remnant ablation is defined as an absence of visible rai uptake on a subsequent diagnostic rai scan or an undetectable stimulated serum thyroglobulin (off - tg) level . Figure 1 presents the dietary iodine intake in both the usual diet and lid periods . Although the patients tried to maintain their lid according to the guideline of less than 100 g / day their mean daily iodine intakes during two - week lid period were more than 100 g . For patient 1, the mean iodine intake level for usual diet and the lid were 317.2 and 117.0 g / day respectively . Patient 2's levels were 125.2 and 110.9 g / day and patient 3's levels were 217.7 and 100.4 g / day . The values in all three patients were reduced after a two - week lid period, but the iodine / creatinine ratio in spot urine exceeded 66.2 g / g (iodide / creatinine), that was the cutoff value for a poorly maintained lid set by kim et al . . All three patients followed five items out of nine food items in the lid guidelines that were using refined salts instead of sea salts and avoiding seaweeds, egg yolk, processed food, and consumed adequate amount of meat and its product, which was less than 120 g / day . Patient 1 and patient 3 also followed three more items that were avoiding fish, milk and dairy products consumption, soybean pastes and soy sauce made with sea salts . Patient 2, however, did not follow three items and consumed fish, milk and dairy products, condiments including sea salts . The food item in the guideline that all three patients did not follow was avoiding salted vegetables, mainly kimchi during the lid . Patient 2 showed the lowest adherence to the lid and the mean iodine intake level during the lid periods was not much different from patient 2's usual intake level . Though serum off - tg levels became undetectable (<1.0 ng / ml) for patients 1 and 2, patient 3's off - tg level was 1.07 ng / ml and all three patients showed visible rai uptake . We evaluated dietary iodine during lid and successful ablation of the rai therapy for three differentiated thyroid cancer patients . All three patients showed low adherence to the lid guideline, in particular for salted vegetable and exhibited remnant thyroid activity in the second post - rai therapy scan . Dietary assessment of iodine intake is challenging because the large day - to - day variation makes it difficult to quantify the " usual " iodine intake . However, the dietary iodine intakes are comparable with the previous findings where dietary iodine intake was reported 478 g / day for korean healthy adults using food frequency questionnaire and 312 g / day in men and 413 g / day in women for japanese using 7-day dietary records . In besides, we measured urinary iodide excretion in both usual diet and the lid periods along with dietary iodine and both two values showed the same trend of being markedly reduced . Although our patients were intensively educated on maintaining the lid, all of their dietary iodine intake levels exceeded the less than 100 g guideline set by the kta . Patient 2 did not follow the lid guidelines such as avoiding dairy, fish and kimchi . Patients 1 and 3 followed kta guidelines except for the restriction on kimchi . Maintaining the lid is challenging for korean patients, whose regular diet consists of foods with very high iodine content, such as seaweeds, seafoods, soy sauce, soy bean pastes, and salted vegetables such as kimchi . According to moon et al ., thyroid cancer patients had considerable knowledge of high iodine content foods but they had a difficulty to prepare low iodine dishes not using sea salts . Though seaweed has the highest iodine content and is by far the biggest contributor to dietary iodine consumption, the patients' main source of iodine was kimchi during the lid period . Kimchi is a main side dish in our korean diet and is usually made of large amounts of sea salts, garlic, red pepper, salted fish and other spices . According to analytical values determined by the korean food and drug administration (kfda), the iodine content per 100 g of radish kimchi, napa cabbage kimchi, and young radish kimchi are 198.4, 143.4, and 107.4 g respectively . Therefore, avoiding foods with a high sea salts content, such as kimchi, is very important for successful rai therapy . Although clinical dietitian put enough emphasis on avoiding sea salts or replacing sea salts with refined salts, it is very challenging for patients to distinguish the dishes made by refined salts from all dishes or to prepare their own dishes with refined salts in daily life . To increase the adherence of the lid, more practical dietary strategy for avoiding sea salts or for replacing sea salts in our korean dishes low adherence to the lid guideline with more than 100 g / d of dietary iodine intake level negatively influenced the efficacy of rai therapy . Future studies are required to explore the influence of various dietary iodine intake levels on the efficacy of rai therapy and to develop more practical guidelines to facilitate the lid maintenance.
First described by cushing in 1898, chyle fistula can induce severe nutritional, metabolic and immune disturbances, delay wound healing, prolong hospital stay and, in the past, even resulted in death [1, 2]. The incidence rate reported in the literature ranges from 0.5 to 2.5% [1, 2, 3, 4, 5, 6, 7, 8]. In thyroid surgery, the rate of chyle fistula increases significantly (p <0.001) with the extent of the surgical procedure: 0.5% after less than total surgery with central compartment node dissection (ccnd), 0.8% in patients with total thyroidectomy and ccnd, 5.1% after total thyroidectomy associated with ipsilateral neck dissection, and 6.2% in patients treated by total thyroidectomy with bilateral neck dissection . Due to anatomic reasons (left side termination of thoracic duct in 7592%), these injuries occur predominantly on the left side in patients undergoing lateral neck dissection [3, 5, 9]. Reported a higher but not significant incidence of chyle leakage on the right side (8.9 vs. 7.5%; p = 0.802). Most chyle fistulas (2575%) develop postoperatively, with a 86% rate of occurrence between the first and the third postoperative day . A 27-year - old woman was referred to our department for the surgical treatment of a goiter with hyperthyroidism treated for 2 years with neomercazole 10 mg / day and levothyrox 50 g / day . Ultrasonography showed a suspicious 11-mm nodule in the left superior lobe and two ipsilateral 20-mm cervical nodes . Fine needle cytology aspiration of cervical lymphadenopathy confirmed the involvement of a papillary thyroid cancer . In may 2010, pathologic specimen examination showed a 12 15 mm papillary cancer in the left superior lobe without extrathyroid extension ., a high - output lymph fistula with milky appearance was observed following a drainage collection of 2,300 ml . We decided to adopt a conservative medical management consisting of a diet of medium - chain triglycerides for 3 days . Persistence of the fistula (1,400 ml / day) on day 6 led to the introduction of a strict diet and parenteral nutrition, followed by a 50% reduction of the daily drainage (700 ml / day) of the chyle fistula . On day 8, after adjunction of a pharmacological treatment with subcutaneous octreotide (0.1 mg every 8 h), the fistulous debit decreased gradually until it disappeared completely after 6 days (fig . Oral nutrition was initiated and the cervical closed suction drains removed . On postoperative day 16, the patient was discharged home in good condition with correct wound healing . One month later, the patient underwent a radioablation therapy (3.56 gbq) after a radioiodine scanning revealed no persistent disease or distant metastases . After a follow - up of 10 months, this young female had no signs of either local and/or distant recurrence or of postoperative functional and cosmetic sequelae . Priority must be given to the prevention of lymph fistulas related to an injury of the main thoracic duct or one of its multiple branches (40% of cases) [3, 6, 8, 10]. A meticulous surgical technique combined with magnifying glasses has recently been advocated by lorenz et al . . Intraoperative diagnosis of lymphatic leakage is better treated immediately by ligature (3 - 0 or 4 - 0 non - absorbable) than suture procedures [6, 9]. In most cases, postoperative diagnosis is clinically obvious and no complementary investigations such as lymphoscintigraphy and/or ct scan are required [3, 8]. Chyle fistula usually starts after an oral diet, with an increase of drainage volume with milky appearance, swelling of supraclavicular fossa, erythema or induration of the skin [3, 6, 8]. Measurement of triglyceride concentrations in postoperative fluid drainage may assist the early diagnosis of chyle fistula . A triglyceride level exceeding 100 mg / dl, a greater serum concentration or a lymphocyte count of 50% are thought to support the diagnosis [4, 6, 7, 8, 9, 11 . Although several therapeutic approaches, including nutritional, surgical and pharmacological procedures, have been proposed, there is currently no clear consensus on the optimal management of thoracic duct fistulas . The decision between conservative and surgical treatment options still remains a subject of debate in literature reviews [5, 6, 7, 8, 11]. Conservative medical management includes several lines of treatment and is imperatively associated with adequate drainage, applying pressure dressing, serial aspirations, bed rest and nutritional modifications [11, 6, 7, 8, 8]. Except for initially large chyle leaks, conservative methods should be considered first since they allow a cure rate ranging from 58 to 100% . The first step is an elemental diet supplemented with medium - chain triglycerides, directly absorbed into the portal circulation by passing the lymphatic system [4, 6, 7, 8, 9]. Conversely, long - chain triglycerides entering the blood stream via the chyle should be avoided [4, 7, 8]. According to lorenz et al ., dietary restriction was less successful than total fasting . In case of failure and despite the increased costs, a total parenteral nutrition is recommended as a second - line approach in most institutions [4, 6, 7, 8]. Furthermore, the optimal timing and the decision between conservative and surgical options remain unclear . For cokun others [1, 4, 8, 9], a persistent drainage output of> 600 ml / day for 57 days despite medical conservative therapy or an extremely high output (> 1.52 liters) may require surgical re - intervention . More recently, lorenz et al . Have suggested a daily 300-ml output drainage threshold and an early decision on day 4 after initiating fasting protocol in favor of either continued medical treatment or surgery . The aim of surgical therapy is to close the leak site with local application of fibrin glue, a pectoralis major muscle flap transfer or an absorbable mesh [6, 7, 8, 9]. En bloc ligature of the thoracic duct by videothoracoscopy has also been proposed in patients who not respond to cervical re - intervention [2, 3, 5, 9, 11]. However, sclerotherapy devices with topical application of tetracycline or doxycycline have been abandoned because of local neurotoxicities (phrenic or vagus nerve palsy) [2, 6, 9]. Somatostatin and its long - acting analog octreotide were first described in 1990 by ulbarri et al . As an effective and successful adjunct to conservative therapy of iatrogenic thoracic duct injuries . These preliminary data were confirmed 10 years later by markham et al . In a prospective study on dogs demonstrating a threefold decrease in thoracic duct fistula output after administration of octreotide . It reduces gastrointestinal chyle production by decreasing splanchnic blood flow and decreasing gastric, biliary, pancreatic and intestinal secretions [1, 2, 4, 7, 11]. Administered at a recommended dose of 3.512 g / kg / h for 314 days, octreotide therapy requires good monitoring of blood glucose levels every 6 h because of the inhibition induced by insulin, glucagon and motilin secretion . The risk of cholecystitis secondary to cholestasis should also not be underestimated in patients eligible for prolonged treatment . In addition to recent data [1, 4, 6, 7, 8, 11, 15], our observation demonstrated that octreotide is a promising alternative therapy useful for decreasing chyle fistula after thyroid cancer surgery and increasing the success of conservative management of this worrying complication . Advances in surgeons knowledge of the anatomic landmarks and variability of the cervical portion of the thoracic duct should minimize the incidence of injuries which lead to an often unrecognized but significant morbidity rate.
Hepatomegaly and elevated liver enzyme levels in type 1 diabetes mellitus (dm) patients can be caused by hepatic glycogenosis, viral hepatitis, autoimmune hepatitis, or metabolic liver diseases . Mauriac syndrome is a rare complication of type 1 dm that is characterized by hepatomegaly due to hepatic glycogenosis, growth retardation, delayed development of puberty, and cushingoid features . It is caused by abnormal hormone secretion and metabolic abnormalities because of inappropriate glycemic control1). If the insulin dosage is insufficient to control hyperglycemia, it can stimulate hepatic glycogenosis and fatty acid synthesis, resulting in inhibition of glycogenolysis and hepatomegaly . Hepatic glycogenosis in type 1 dm patients is a reversible complication by strict blood glucose level control, which is different from nonalcoholic steatohepatitis (nash) in obese type 2 dm . Thus far, eight cases of type 1 dm accompanied by hepatic glycogenosis and three cases of mauriac syndrome have been reported in korea234). We report a case of hepatic glycogenosis mimicking mauriac syndrome in a patient with poorly controlled type 1 dm who showed hepatomegaly, cushingoid face, mild growth retardation, and delayed menarche . A 14-year - old girl was admitted to the catholic university of korea, seoul st . Total daily insulin dosage was 12 iu (0.26 iu / kg / day). She had previously been admitted seven times because of diabetic ketoacidosis in the previous 2 years . At the time of admission, the patient had clear consciousness, with the following vital signs: blood pressure, 90/60 mmhg; pulse, 118/min; and body temperature, 36.5. the height of the patient was 155 cm, and her weight was 46 kg, which were in the 25th and 50th percentiles, respectively . Growth velocity decreased to 3.3 cm / yr in the past 2 years (fig . The patient presented with tanner stage iv breast and tanner stage iii pubic hair; however, the patient did not experienced menarche . Bone age was delayed by 2 years compared to her chronological age . Upon physical examination blood test results revealed the following values: white blood cells, 4,840/mm (47.5% neutrophils and 42.6% lymphocytes); hemoglobin, 13.4 g / dl; platelets, 218,000/mm; aspartate aminotransferase (ast), 1,408 iu / l; and alanine aminotransferase (alt), 459 iu / l; gamma glutamyltransferase, 430 iu / l; and amylase, 115 iu / l . The glycosylated hemoglobin (hba1c) level was 14.3%, and ketone was not detected in urine . To rule out viral hepatitis, we performed serologic tests for hepatitis a, b, and c; epstein - barr virus; and cytomegalovirus infections, and obtained negative results . Furthermore, the results of autoimmune hepatitis, including antinuclear antibody, antismooth muscle antibody, and antiliver kidney microsome antibody, were all negative . Thyroid function was normal and the following hormone levels were presented: insulin - like growth factor-1 (igf-1), 203 ng / ml; luteinizing hormone (lh), 4.47 miu / ml; follicle - stimulating hormone (fsh), 4.77 miu / ml; and estradiol (e2), 35.44 pg / ml . The plasma cortisol level was 5.73 g / dl and the adrenocorticotropic hormone level was 17.14 pg / ml at 8:00 am . 3). Through a liver biopsy, we found ballooning degeneration with nuclear vacuolization of hepatocytes (fig . Hepatocytes were strongly positive in a periodic acid - schiff (pas) stain (fig . 4b), and such positive finding disappeared with diastase (d - pas) (fig . The insulin dosage was gradually increased over 3 months; thus, the total daily insulin dosage became 36 iu (0.65 iu / kg / day). To prevent hypoglycemia, we prescribed 60 g (1.3 g / kg) of uncooked cornstarch between meals and bedtime . After implementing strict glycemic control, hepatomegaly was improved and elevated transaminase levels returned to normal ranges . Moreover, the patient manifested menarche at the age of 15.3 years and a regular menstruation cycle thereafter . Hepatic glycogenosis in type 1 dm is a key mechanism of mauriac syndrome and is a rare diabetic complication first reported by mauriac in 19305). Hepatic glycogenosis can be caused by either a persistent hyperglycemia due to insulin deficiency or conversion of excess glucose administered to control hypoglycemia due to excessive insulin administration . With regard to the latter mechanism, furthermore, the secretion of the antagonistic hormone cortisol increases in type 1 dm patients with poor glycemic control . Such excess cortisol inhibits hepatic glycogenolysis and increases fatty acid synthesis in the liver, causing hepatomegaly . Moreover, hypercortisolism can result in cushingoid features and delay in bone maturation, which in turn leads to delayed growth and puberty1). Growth retardation and delayed puberty with hepatic glycogenosis in uncontrolled type 1 dm patients are distinct characteristics of mauriac syndrome . In patients with mauriac syndrome, circulating igf-1 levels are normal or reduced despite normal hypothalamic - pituitary functions . Patients with mauriac syndrome also exhibit resistance to growth hormones, which is caused by a decrease in igf-1 bioactivity, igf-1 receptor defects, and circulating igf-1 inhibitors9101112). Delayed puberty can manifest as a result of insulin deficiency, hyperglycemia itself, or decreased lh and fsh secretion caused by the gonadotropin - releasing hormone due to stress hormone secretion after poor glycemic control . Insulin administered to the subcutaneous tissue bypasses the hepatic first - pass metabolism and is absorbed directly via systemic circulation . Then, the insulin binds with the receptor at the ovary to excessively stimulate the ovary, thereby increasing androgen secretion . If peripheral insulin resistance increases because of hyperglycemia, polycystic ovary syndrome can occur, causing ovarian failure and amenorrhea1314). Although growth retardation and delayed puberty were not prominent in our patient, the patient exhibited very similar clinical manifestations of mauriac syndrome . The reason that the signs of growth retardation and delayed puberty were mild in our patient was probably because hepatic glycogenosis occurred in the late stage of pubertal development, thereby only minimally affecting the patient's growth and puberty . As we did not measure 24-hour urinary free cortisol levels and the 8:00 am . If the symptoms of mauriac syndrome recur, a detailed evaluation of hypercortisolism will be necessary . Treatments of hepatic glycogenosis in type 1 dm include strict glycemic control through appropriate diet and insulin dosage . Although regular diet and sufficient insulin dosage are important in controlling hyperglycemia, determining the appropriate insulin dosage is difficult when hyperglycemia is accompanied by frequent hypoglycemia, as in the case of our patient . As glycogenolysis does not properly occur even with hypoglycemia in the patient with hepatic glycogenosis, additional glucose supply is necessary . However, the glycemic effects of conventional snacks only manifest 2 hours after consumption . Moreover, as the glycemic effect inevitably decreases with time, hypoglycemia can recur . Consequently, consuming uncooked cornstarch to induce delayed glycemic effects facilitates the prevention of hypoglycemia15). Hepatic glycogenosis does not occur in all patients with type 1 dm who have poor glycemic control . Although the risk factors of hepatic glycogenosis are not yet known, we postulate that wide fluctuations of blood glucose levels and a long period of poor glycemic control increase the risk of hepatic glycogenosis . However, hepatic glycogenosis in type 1 dm is a reversible complication that is different from nash, which is often presented in obese type 2 dm patients . Therefore, appropriate glycemic control is important to the treatment and prevention of hepatic glycogenosis in type 1 dm.
An inflammatory myofibroblastic tumor (imt) is a distinctive neoplasm composed of myofibroblastic and fibroblastic spindle cells accompanied by inflammatory infiltration of plasma cells, lymphocytes, and eosinophils . It is important to distinguish this tumor from other malignant spindle cell tumors, such as the sarcomatoid variant of urothelial carcinoma and leiomyosarcoma . We report a case of imt of the urinary bladder in a 52-year - old male diagnosed by transurethral resection of the bladder tumor (turbt) treated by partial cystectomy . His medical history revealed a diagnosis of rheumatoid arthritis 3 years ago, and prednisolone 10 mg / day was subsequently prescribed . Cystoscopy revealed a solitary nonpapillary tumor with surrounding edema at the dome of the bladder (fig . 1a). Enhanced computed tomography (ct) and magnetic resonance imaging (mri) revealed an early enhancing bladder tumor infiltrating the outside of the bladder muscle layer (fig . Intraoperative findings showed a large solid bladder mass, measuring approximately 3 cm from the dome to the anterior wall of the bladder . We resected the tumor to the depth of the muscle layer, but normal muscle tissue was not seen . Histopathology results were consistent with an imt . The bladder tissue, including muscle, was widely infiltrated by spindle cells in a myxoid stroma accompanied by infiltration of inflammatory cells (fig . Immunohistochemical staining was positive for vimentin and smooth muscle actin (fig . 2c) and negative for ae1/ae3, desmin, myogenin, s-100, cd34, c - kit, cd68, and bcl-2 . The excised specimen is shown in figure 2d . A pathological examination of the excised specimen revealed the proliferation of spindle cells expanding into the bladder musculature, accompanied by inflammatory cell infiltration . For the assessment of therapeutic options, we measured the expressions of anaplastic lymphoma kinase (alk), vascular endothelial growth factor (vegf), and cyclooxygenase 2 (cox2), known as growth factors involved in tumor proliferation . It is characterized by atypical spindle cell proliferation and inflammatory cell infiltrates primarily involving lymphocytes and plasma cells . Although it has been associated with trauma, surgery, and infection, the majority of imt cases occur spontaneously . Imt tumors are classified as intermediate (rarely metastasizing) tumors according to the who classification of soft tissue tumors . Imts occur in the mesentery, omentum, retroperitoneum, pelvis, and abdominal soft tissues in 73% of cases . However, the occurrence of an imt in the urinary bladder is unusual . A systematic review by teoh et al . Evaluated 182 imt cases and found a mean age of patients of 38.9 years, with a predilection for females . Imts resemble malignant spindle cell tumors, such as sarcomatoid carcinoma, leiomyosarcoma, or rhabdomyosarcoma, making diagnosis difficult . Recent reports have indicated that alk, which was originally identified as a protein overexpressed in anaplastic large - cell lymphomas, was also overexpressed in a substantial proportion of imts [5, 6]. A positive finding of alk by immunohistochemistry in up to 87.5% of imts can be useful for the differentiation of imts from other spindle cell tumors . In this case, alk gene translocation in imts has also been reported, for which detection by fluorescence in situ hybridization (fish) is useful . However, performing fish in paraffin - embedded specimens is questionable for obtaining a precise diagnosis . Alk protein expression, as determined by immunohistochemistry, correlates well with the presence of a rearrangement in the alk gene [7, 8]. Therefore, we did not undertaken fish analysis in this case because of the strong immunohistochemical positivity of alk . In the consideration of a therapy for imt, surgical resection, most patients underwent turbt (60.8%); others had partial (29.2%) and radical cystectomy (9.2%), and 5 patients experienced local recurrence . While partial or radical cystectomy ensures complete resection of imt, turbt is also a considerable choice, given the benign course of an imt . In the present case, ct, mri, and the examination of a turbt indicated a muscle layer - infiltrating lesion, so we performed a partial cystectomy . There were no reports as to the safety margin of imt in the bladder at partial cystectomy . We performed the operation by setting a margin of about 1 cm, and there was no sign of local recurrence in the bladder wall . For performing the operation while keeping the bladder capacity, further investigation as to the surgical margin cox2 and vegf expression have been detected in imts and are thought to be therapeutic targets . Additionally, as an anti - inflammatory drug, cox2 inhibitors used for nonresectable imts have often been reported . An alk inhibitor, crizotinib, has also been used in the treatment of imts . In the present case, we examined cox2, vegf, and alk protein expression with immunohistochemistry and found imt tissues to be vegf and alk positive . Prednisolone has previously been prescribed in this case . We also thought that the tumor was resectable by partial cystectomy and therefore decided against a course of pharmacotherapy . However, in the case of recurrence when the tumor is not resectable, pharmacotherapy may become a viable therapeutic option . In conclusion, a typical imt can be locally aggressive and may require radical surgical resection; close follow - up is therefore warranted . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written informed consent is available for review from the editor - in - chief of this journal.
Ultrasound - guided fine - needle aspiration (fna) has become the gold standard for differentiating benign and malignant thyroid nodules due to the suboptimal accuracy using gray scale features alone . Besides for mild associated pain, development of postprocedural hematoma has been reported from 1.9% to 6.4%, with seven reported cases of life - threatening hematomas relating to airway obstruction . The true rate of complications following thyroid nodule fna is likely much higher though as most go undocumented and unpublished . Although fna plays an essential role in thyroid nodule evaluation, the potential for a nondiagnostic (nd) specimen can lead to repeat procedures, increasing patient morbidity, cost, chance for complication, and delay in diagnosis . Recently, thyroid nodule fna pathological reporting has been standardized based on the bethesda system for reporting thyroid cytopathology adopted by the american society for clinical pathology (ascp). Based on their criteria, an adequate specimen needs to contain at least six groups of follicular cells with each group composed of at least 10 cells each . An nd specimen can be secondary to obscuring blood, overlying thick smears, air drying of alcohol - fixed smears, or inadequate number of follicular cells . According to the ascp, nd specimens occur in 2%20% of cases but ideally should be limited to no more than 10% of all thyroid nodule fnas . Recently, the use of ultrasound strain elastography (use) has emerged as a promising diagnostic tool in differentiating benign and malignant thyroid . A novel approach aimed at decreasing the rate of nd nodule fna maybe to use elastography as a map, targeting biopsy needles to areas of lower strain (i.e., stiffer). Several papers have been published illustrating increased detection of prostate cancer using use - targeted interventions through a transrectal approach . Our study was performed to evaluate the role of use - targeted fna in decreasing the rates of nd aspirates . We performed a review of all thyroid nodule fna performed over a 3-year period comparing fna results when an elastogram was available before fna or not . Our hypothesis is that the presence of an elastogram before fna would decrease the rate of nd specimens . The study was health insurance portability and accountability compliant, and informed consent was waived . Between january 2011 and january 2014, a total of 225 patients underwent ultrasound - guided thyroid nodule fna . Among them were 171 females and 54 males with ages ranging from 27 to 90 . The demographical data recorded included patients' age and gender as well as which thyroid lobe contained the nodule biopsied [table 1]. Gray scale nodule characteristics that were recorded included the presence of microcalcifications, vascularity, solid components, and nodule size [table 1]. The decision to perform use was based strictly on suspicious gray scale characteristics, and guidelines set forth by the society of radiologists in ultrasound . Patient demographics and nodule characteristics during this period, use for thyroid nodules was performed in cases where the available technician was comfortable and experienced with the elastography software . When elastography was performed, the applied transducer pressure was standardized by a real - time analysis utilizing a scale that was displayed at the right upper corner of the elastogram image [figure 1]. The nodule strain elastogram was displayed using a 256-color mapping scale [figure 2]. The degree of strain was assigned a given color shade utilizing a range from red (greatest strain, soft) to green (intermediate strain) to blue (least strain, hardest component). Real - time applied transducer pressure analyzed by numeric scale displayed at the right upper corner of the elastogram image (red box). Nodule strain elastogram is displayed by a 256-color mapping scale with red representing the softest components and blue representing the hardest components . We separated patients into two groups, based if an elastogram was available for the radiologist to review before fna or not . All fna were completed by one of two fellowship - trained radiologists with specialty in ultrasound - guided procedures . A 21-gauge needle was used in every case, and aspiration was aided by a 5-cc syringe . All pathological analyses were performed by one of three board - certified cytopathologists . In all cases, the final pathological diagnosis was reviewed through the institution's electronic medical record for every case . The chi - square test was used to determine statistically significant variances in the demographic parameters . There was no significant difference between the two groups in demographics (including gender, age, and which thyroid gland contained the nodule) as well as nodule characteristics . Regarding the specimen adequacy, the total percentage of nd aspirates was 12.2%, slightly higher than the 10% supported by the ascp . In analyzing the two groups, when an elastogram was available before fna, the nd rate was 10% (14/140) which was in concordance with the ascp recommendations . However, the group without an elastogram available had an nd rate of 16% (13/81). However, this difference was not found to be statistically significant, p = 0.205 . Thyroid nodules continue to pose a diagnostic dilemma secondary to poor sensitivity and specificity using gray scale features alone . Although considered the gold standard for nodule evaluation, fna carries its own risks including patient pain, cost, and possible complications (although rare). The need for repeat fna secondary to nd results can cause further patient harm as well as delay time to treatment and increase patient anxiety . To help increase the rate of adequate specimen recovery, the use of ultrasound to guide fna has become the standard of care . With the assistance of ultrasound - guided fna, the rates of nd specimen aspirates still range up to 20% . In a recent meta - analysis, even the addition of rapid onsite evaluation by a cytopathologist has shown considerable variability in helping to reduce nd rates . Ex vivo studies have found that malignant thyroid lesions have a stiffer architecture compared to benign thyroid stroma . First described in 1991 by ophir, tissue stiffness could be measured by applying an external axial stimulus and viewing the associated compressibility . The continued research in elastography has focused on its ability to distinguish benign and malignant lesions by virtual palpation . Several studies have been performed to evaluate the role of elastography in targeting interventions . Most papers have focused on ultrasound - guided transrectal biopsies of the prostate gland . Using elastograms, small studies have been able to show enhanced detection rates as well as decreasing the number of core specimens required for diagnosis . To date, there has been only one study performed evaluating strain elastography - guided fna of thyroid nodules . In 2013, yildrim et al ., prospectively analyzed the rates of nd aspirates between specimens obtained from regions of higher strain and lower strain from 96 patients . The authors found a significant improvement in specimen adequacy when aspirates were obtained from regions that demonstrate a stiff elastogram as described previously . Our study was aimed at replicating yildrim's results in a larger patient sample and hoping to show an improved rate of nd aspirates . In our retrospective review, although there was a decreased rate of nd specimens in the group that use was performed (16% vs. 10%), this was not found to be statistically significant . First of all, we performed a retrospective review, which by its very nature allows for confounding bias . Furthermore, although an elastogram was available for the radiologist before fna in the elastography group, we were unable to prove that stiffer regions were actually targeted and aspirated during the procedure . Furthermore, it is possible that stiffer regions of nodules were sampled in the group without elastograms even though elastography was not performed . Finally, as our study was a retrospective review, the pathologist performing the specimen analysis was not controlled for, leading to possible variability . Another possibility exists to explain why the presence of an elastogram prior the fna did not significantly reduce the rate of nd results . Our study was predicated on the idea that stiffer regions within nodules would likely decrease nd aspirates as these regions may represent more cellular areas . However, the development of a stiff consistency in many cancers is secondary to a desmoplastic transformation that takes place . This process is composed of activated fibroblasts (also known as myofibroblasts), tumor vessels, inflammatory cells, and extracellular matrix components . Activated carcinoma induced fibroblasts, along with other extracellular matrix proteins, comprise a dominant proportion of several carcinomas, including more than 90% of overall tumor mass in pancreatic cancers . It has even been shown that the complex relationship between these components may promote tumor cell growth . It is possible that the stiffer regions within thyroid nodules are more a product of fibroblastic growth than increased cellular density . We were unable to demonstrate a significant difference in thyroid nodule aspirate adequacy between patients who had an elastogram present before fna compared with those without one . These results may be secondary to lack of power, poor study design versus fibroblastic growth as described above . We hope our study raises awareness of the exciting prospect of use - targeted biopsy and springs future studies confirming its potential.
Molecular hydrogen (h2) is a zero - emission energy carrier and is one of the best alternative fuels for the future, especially in urban areas with stricter environmental requirements . Hydrogenase (h2ase) enzymes catalyze the bidirectional reaction of 2h + 2e h2 (jugder et al ., 2013; kim & cha, 2013) and thus have attracted interest for potential applications in h2 production in addition to their importance in biological sciences (mertens & liese, 2004; cammack et al ., 2001; heinekey, 2009; tard & pickett, 2009; yang et al ., 2011; fritsch et al ., 2013; simmons & artero, 2013; matsumoto et al ., 2013). While both diiron ([fefe]) and nickel iron ([nife]) h2ases possess high catalytic activity (evans et al ., 2013; armstrong, 2009; shafaat et al ., 2013; lubitz et al ., 2014), the latter are attractive practically since they exhibit greater o2-tolerance (guiral et al ., 2006; evans et al ., 2013; lauterbach et al ., 2015 [nife] h2ase contains several fe s clusters and one nife active site, in which fe is coordinated by one co and two cn ligands (kamali et al . The ni is coordinated to the protein matrix by four cysteinyl thiolates (s), two of which serve as bridging ligands to fe . In addition to these two s ligands, there could be a third ligand to bridge the two metal atoms in several enzymatic states; for example, a hydroxide ligand in the oxidized inactive forms (ni a and ni b) (dole et al ., 1997; gu et al ., 2003; gastel et al ., 2015) and a possible hydride (ni h fe) in the active forms ni c and ni 1(b 1)1(b 6)], and the possible mechanism for h2 binding and cleavage at the h2ases active site . For example, via a density function theory (dft) calculation, the structures in figs . 1(b 5) and 1(b 6) are believed to be the most likely candidates in the real ni information about the mechanism in turn could be useful for better producing h2 in the future . Despite progress in characterization of h2ases by crystallography, infrared (ir) spectroscopy (lubitz et al ., 2014; fontecilla - camps et al ., 2007; de lacey et al ., 2007) and other x - ray spectroscopies (wang et al ., 2000; wang, patil, gu et al ., 2001; wang, patil, ralston et al ., 2001), questions remain about the detailed molecular and electronic structure of various intermediates and inhibited species . These highly debated questions include the nature or even the existence of a hydride bridge (ni h fe) in ni recent high - resolution crystallography demonstrated that the electron density as a hydride was detected at the bridging position between ni and fe (ogata, nishikawa & lubitz, 2015). R represents a special challenge for spectroscopic studies for a number of reasons: (i) it is epr (electron paramagnetic resonance) silent; (ii) the raman spectroscopy presents a problem due to its photoreactivity; and (iii) ir spectroscopy for hydride bands is extremely difficult to observe (jayapal et al . Thus there has been no progress in traditional vibrational spectroscopy so far and the existence of ni h fe in ni nuclear resonance vibrational spectroscopy (nrvs) scans an extremely monochromatic (1 mev) x - ray beam through the nuclear resonance (at 14.4 kev for fe), and measures the corresponding creation or annihilation of phonons (seto et al ., 1995; sturhahn et al . It is a relatively new x - ray spectroscopy that became available due to the development of third - generation synchrotron sources and advanced x - ray optics . It has several distinguished advantages in comparison with traditional vibrational spectroscopic techniques such as ir and raman spectroscopies (smith et al ., 2005; kamali et al ., 2013; pelmenschikov et al ., 2011). In the past ten years, this technique has revealed / resolved fe s / p / cl and fe co / cn / no vibrational modes inside various inorganic complexes and iron enzymes and has become an excellent pin - point tool in recent years to study iron - specific inorganic and bioinorganic systems (smith et al ., 2005; cramer et al ., 2007; tinberg et al ., 2010 in addition, fe h - related nrvs features have also been resolved in several inorganic complexes (bergmann et al . Via a series of careful and strenuous measurements, we have revealed the first direct spectroscopic evidence for ni r form of desulfovibrio vulgaris miyazaki f (or dvmf) [nife] h2ase using fe - specific nrvs . While its biochemical science and theoretical simulations have been presented and discussed recently (ogata, kramer et al ., 2015), the measurement details, especially the strenuous experimental journey leading to the successful observation of the weak ni fe in a [nife] h2ase sample, and the task will face great difficulties due to the following two aspects: (i) a fe h - related bending or wagging mode has a much weaker nrvs signal than fe co (pelmenschikov et al ., 2011) while fe co in nife h2ase is already rather weak (kamali et al ., 2013; ogata, kramer et al ., 2015 (ii) as will be discussed in detail later, its real energy position is far from either model complexes or theoretical predictions and was virtually unknown before it was observed experimentally . This article evaluates all the matters related to whether the feature is observable, where to search for a fe h - related feature, whether the observation is reliable, and how the experiments identify the observed 675 cm peak as a fe h - related feature . R nrvs spectra were recorded at spring-8 beamlines bl09xu (yoda et al ., 2001) and bl19lxu (yabashi et al . Bl09xu is a dedicated nuclear scattering beamline, and its instrumentation and properties have been discussed in detail elsewhere (yoda et al ., it has a si(111) double - crystal high - heat - load monochromator (hhlm) producing 14.4 kev radiation with 1.0 ev resolution, followed by a high - energy - resolution monochromator (hrm) [ge(422) 2si(975)] producing 14.4 kev radiation with 0.8 mev resolution . The beam flux was 1.4 10 photons s at 0.8 mev energy resolution . The beam size was about 0.6 mm (height) 1 mm (width). Bl09xu has a dedicated nrvs measurement system, including a 2 2 avalanche photodiode (apd) detector array, a set of data - acquisition electronics and a control computer with data - acquisition software . It also has a dedicated liquid - helium (lhe) flow cryostat for measuring nrvs at a cryogenic temperature for air- or temperature - sensitive samples, such as h2ases . However, due to the extremely short distance from the samples surface to the apd array, a cold - finger cryostat has to be used instead of a more efficient gas exchange cryostat (dong et al . The real sample temperatures were much higher than 69 k, e.g. 5070 k (wang et al ., 2012). This standard nrvs setup has been used in many nrvs measurements in the past and their details are discussed elsewhere (cramer et al ., 2001) provides the fundamental beam in the 7.218 kev energy region, covering fe nuclear resonance at 14.4 kev . As shown in fig . 2(a), it has a 25 m - long undulator (hara et al ., 2002) instead of the usual 5 m - long undulator at spring-8 . Therefore its hhlm provides 2.5 10 photons s at 14.4 kev, i.e. About five times higher than the beam intensity at bl09xu (5 10 photons s after hhlm). This beamline has many advanced applications and publications over a broad area from time - resolved measurements to weak feature observations (tanaka et al . It is also a good choice for nrvs measurements on weak vibrational features, which requires high beam intensity . However, bl19lxu is not a dedicated nuclear scattering beamline and it does not have a fixed hrm and nrvs measurement station . 2b) which consists of a hrm, a lhe cryostat, a 2 2 apd detector array, a rack of associated electronics, and a control computer with data - acquisition software . While these items are duplicates of those used at bl09xu, they need to be integrated into bl19lxu s experimental hutch 1 within about 36 h (including optimization time). Therefore, bl19lxu has similar hhlm and hrm as those at bl09xu, and provided 14.4 kev radiation with 0.8 mev resolution, which is suitable for nrvs measurement . The beam size was also 0.6 mm (height) 1 mm (width), the same as that at bl09xu . After the hrm, a maximum 4.2 higher beam flux was recorded in comparison with that for bl09xu . However, owing to various practical limitations as well as using a moved - in hrm and nrvs apparatus, the ratio in nrvs counts per second (cts / s) for the two beamlines is 2.53.4 instead, while for bl09xu the cts / s is stable . For example, in comparison with the bl09xu standard, the bl19lxu beam time for measuring the two ni r in h2/h2o (nir - h for short) samples in this study has a bl19/bl09 cts / s ratio of 2.6, while that for measuring the ni r in d2/d2o (nir - d for short) has a ratio of 3 ., 2007; seto et al ., 1995; sturhahn et al ., 1995; yoda et al ., 2001), with a step size of 0.28 mev at bl09xu and 0.27 mev at bl19lxu . The difference is due to minor difference in energy scales for the different beamlines; the raw step sizes before the energy calibrations were both 0.295 mev . The 14.4 kev beam with an energy resolution of 0.8 mev was scanned through the defined energy region; the delayed nuclear fluorescence and fe k fluorescence from fe were detected with the 2 2 apd array, processed by the electronics, and stored / displayed via the control computer . The scanning region covered from 30 mev (240 cm) to 70125 mev (5601000 cm) depending on the different samples used and the different search cases ., 1995), where the observed raw nrvs spectra (cts) were calibrated to the nuclear resonance peak position, normalized to i 0, summed and converted to the single - phonon fe partial vibrational density of states (pvdos for abbreviation). The spectral conversion was optimized when the observed stokes / anti - stokes imbalance matched the imbalance calculated using the entered temperature as a parameter . The real sample temperatures were thus obtainable with the measured nrvs s stokes / anti - stokes imbalance (wang et al ., 2012). The energy position for each scan was calibrated by aligning the elastic peak to zero during the nrvs spectral analysis with phoenix . The energy scales were usually calibrated with a standard sample of [fecl4][net4] with a prominent peak at 380 cm . Alternatively, the energy scales can also be calibrated with fe metal powder at the quick - switch calibration stage at the back of the main measurement stage (wang et al . It has a clear peak at 287 cm at room temperature . The background noise level (dark current cts / s) was estimated prior to (and sometimes during) each beam time by tuning the hrm s energy position to about 100 mev or about 800 cm (in reference to the resonance elastic energy), where no vibrational peak is present . Signal was accumulated for either 500 s in total or five consecutive 100 s to obtain an average cts / s . For almost all the beam times, the dark current was 0.03 cts / s for our nrvs measurement system at bl09xu and at bl19lxu . Since the relative strength of nrvs transitions varies dramatically and since it is necessary to emphasize one region of interest (e.g. For searching for ni fe), the scans were divided into segments with different data collection times (seconds per point, or s / p). In general, 13 s / p was used for the range from 240 to 400 cm (covering the fe s region), then 510 s / p for the fe cn and fe co region from 400 to 620 cm . A longer scanning time (1030 s) was used for the candidate ni h fe searching region (e.g. At 620770 cm). This practice is used for the nrvs measurements at both bl09xu and bl19lxu, but the exact timing for different beam times varied a little . The scan details will be discussed again in the results and discussions section . To compare the nrvs data from bl09xu and bl19lxu, we re - scale the bl19lxu counting time based on its maximum cts / s versus the standard maximum cts / s at bl09xu and create bl09xu equivalent seconds, e.g 10 s / p at bl19lxu corresponds to 26 or 30 equivalent s / p at bl09xu [corresponding to a cts / s ratio of 2.6 for measuring nir - h and 3 for measuring nir - d]. Nir - h was measured once at bl09xu and twice at bl19lxu with an equivalent bl09xu time of 10 680 = 680 s / p, 20 24 2.6 = 1248 s / p and 15 12 2.6 = 468 s / p, respectively, in total 2396 s / p (with a percentile of 28%, 52% and 20%). Nir - d was measured three times at bl09xu and once at bl19lxu, equivalent to 3980 bl09xu s / p [= (40 20) + (30 30) + (36 30) + 20 20 3 = 2780 + 400 3 = 3980] in the same region . More s / p was used for nir - d because more time should be used to conclude that there is no signal than to find a weak signal . Fermentation was carried out in a 10 l glass fermenter under anaerobic conditions, and the [nife] h2ase expressed was isolated and purified as described earlier (ogata et al . For preparing nir - h (nir - d), the as - isolated [nife] h2ase was transferred from 25 mm tris - hcl (ph = 7.4) buffer to 100 mm mes (ph = 5.0 or pd = 5.0) and was then purged with 1.2 bar h2 (or 1.3 bar d2) for more than 8 h. the solutions were then loaded into nrvs cells under anaerobic conditions . Fourier transform ir (ftir) spectra were recorded on a bruker ifs66v / s ftir spectrometer to ensure the samples were in their supposed states (ni r1: one of the subforms of ni it was measured in transmission mode in a sealed ir cell at room temperature and with a 2 cm energy resolution . Nrvs or the fe h / d - related bending positions for several fe, 2011; crossland et al ., 2009; schilter et al ., 2012): (i) [feh(d)6][mgbr(thf)2]4 (or fefeh6/d6 for short); (ii) fe(h / d)(co)(dppe)2, dppe = 1,2-bis(diphenylphosphino)ethane (or h / dfeco); (iii) trans-[fe(dmeoprpe)2(n2)h] {dmeoprpe = 1,2-bis[bis(methoxypropyl)phosphino]ethane} (or hfen2); (iv) [(,k2-bdt - h)(-pph2)(-h)fe2(co)5][otf] (or fehfe); (v) [(dppe)ni(-pdt)(-h)fe(co)2(ph3p)] (* = nature abundant, dppe = 1,2-ph2pch2ch2pph2, pdt = sch2ch2ch2s) (or nihfex); (vi) [(dppe)ni(-pdt)(-h / d)fe(co)3] (or nih / dfe). In addition, [fecl4][net4] and metal fe were used as calibration samples for energy scales (smith et al ., 2005; the overall nrvs spectra for dvmf nir - h (blue) and nir - d (red) are presented in fig . The nrvs spectra include the very low energy intensities from the backbone motion, the 100400 cm peaks for fe s cluster vibrations, the 420530 cm peaks for fe cn modes, the 530630 cm peaks for fe co modes (lauterbach et al ., 2015; kamali et al ., 2013) and a weak but clear peak at 675 cm, which are assigned to the ni h 3, the top bars indicate different vibrational regions while the bottom bars illustrate the total time (s) used to measure the nir - h (blue text) and nir - d (red text) in each region . The blue text in the middle shows the signal level (cts / s) for each of the various signature peaks . While these middle blue numbers illustrate the signal level (cts / s) for nir - h, both nir - h and nir - d have about the same signal level . Co / cn features have a much lower cts / s than the fe s features while the ni h fe has an even lower cts / s in comparison with the weak fe this provides readers with a basic idea of how weak the ni h fe peak in ni 1(b 1)1(b 6)] (ogata, kramer et al ., 2015), the existence of ni h fe at 675 cm for nir - h (blue) and its absence for nir - d (red) provided a critical observation reference for the dft calculations, not the other way around . Without this successful observation, r is observable or not . As per the nrvs statistics at bl09xu between 2006 and 2010, when the hrm s energy resolution is at 0.9 mev, 1 mm of fe sample produced a 30 cts / s signal in the elastic peak (wang et al . 4, using 30 cts / s per 1 mm fe . The first two columns describe vibrational feature names and their vibrational energy positions, and the third column presents the nrvs signals for a hypothetical 1 mm h2ase (or 12 mm fe) sample, which is similar to the h2ase measured in the past (kamali et al . It has 360 cts / s in the resonance peak, 0.71.2 cts / s for the fe s peaks (at 150380 cm), and 0.06 cts / s signal for fe co (at 600 cm). Co features were successfully observed with a 1 mm h2ase sample in the past (kamali et al . Fe was too weak to be observed under previous conditions (1 mm h2ase concentration), so we have to estimate its potential cts / s (the numbers below the fe and fe co in available complexes, e.g. 1/5 for nihfe (fig . 5b). A value of 0.012 cts / s is estimated for ni h fe in 1 mm nir - h at bl09xu; it is under the dark cts / s level (0.03 cts / s) and is not observable under the given circumstances . Since the completion of the nrvs measurements for kamali et al.s publication (kamali et al ., 2013), further progress has been made to increase the potential nrvs signal level (cts / s). The major advance is the samples concentration increasing from 1 mm to 4 mm, which puts the ni fe feature at a hypothetical level of 0.012 4 = 0.048 cts / s . Some other minor improvements also contribute to the increase of cts / s: (i) bl09xu s hrm advanced from 0.9 mev to 0.8 mev energy resolution with almost the same level of beam intensity, which led to an increase in peaks cts / s [maximum = (0.9/0.8) = 112.5%]; (ii) due to better control of the sample temperature during the nrvs measurement (wang et al ., 2012), we are able to move the sample even closer to the apd . It is difficult to estimate the real movement of the sample position but we obtained about 10% signal level increase in cts / s due to sample position alternation while still maintaining the sample temperature below 70 k. these two points thus mean all the nrvs peaks have 1.2 height . Fe is then about 0.048 1.2 = 0.058 cts / s, which is comparable with the fe co feature for 1 mm h2ase in the previous measurement (kamali et al . 4 (not counting the narrow columns showing the arrow symbols) shows the estimated cts / s for a hypothetical 4 mm h2ase, which will be measured at the improved bl09xu (1.2). Then, how much time is needed to reveal such a weak peak (0.058 cts / s) with a reasonable signal - to - noise ratio (s / n)? In a previous publication (wang et al ., 2014) we have concluded that a 10 mm fe sample has 1 cts / s in the fe s region and 100 s / p is thus enough for a s / n = 100 = 10 . Using the same principle fe can lead to 0.058 500 = 29 pure cts and s / n = 29 = 5.4 . Although dark - current cts / s limits the best possible s / n, as will be discussed in detail later, an as - high - as - possible statistical s / n is still required and thus 500 s / p is a kind of realistic minimum time required to achieve a reasonable s / n for a weak feature like ni h fe . As a reference, the very weak fe co features for the oxidized and the reduced 1 mm h2ase were observed with 580 and 2180 s / p total scanning time at bl09xu in the previous publication (kamali et al ., 2013). The 500 s / p scanning time means, for example, 25 20 s / p, leading to about 24 h or more beam time for one measurement with one ni fe signal is as important as (if not more important than) knowing it is observable . R, which requires long (e.g. 24 h or more) beam times for each search . Since no nrvs features for iron hydrides or iron deuterides in a real iron enzyme have been published before this work, we used nrvs for a series of iron hydride / deuteride complexes and their fe h - related vibrational positions (fig . 5) as a starting point to discuss the possible location of the ni the nrvs feh6 (blue)/fed6 (red) spectra were among the first nrvs spectra obtained for chemical complexes (bergmann et al . Their features include: the x fe h / x fe d bending modes at 790/572 cm and the fe later on, many other iron - hydride - containing complexes were published or evaluated, e.g. The (h / d)feco model complexes have the x fe h / x fe d at 740/620 cm, respectively (pelmenschikov et al ., 2011). We noticed that the x fe d bending peak for this complex is close to the fe the nihfe complex is used to model [nife] h2ases active site and it mimics the structure for the [nife] center with very minor structural differences (barton et al ., 2009; shafaat et al . Its ni h fe wag mode is at 758 cm (fig . D stretching (mixed with fe) per dft calculation (ogata, kramer et al . It is not fe d - related bending, which is mixed into the fe co features . The nrvs feature most relevant to ni r s ni h fe is the x fe h or ni h fe feature in various complexes . The energy positions of these fe h - related bending modes for feh6, hfeco, hfen2, fehfe, nihfex and nihfe (refer to 2 for the full names of these complexes) are illustrated in fig . 5(b). The blue shaded area highlights the possible fe h - related bending energies while the orange (purple) area indicates the fe d bending (stretching) mode regions . From these fe h / d complexes we have learned the following: (i) although fe d - related bending has more intensity than fe h - related bending, it may mix with fe co and become unresolvable . Therefore, for fe co - containing complexes, the fe h - related bending mode has the highest observable nrvs peak among all of the fe h / d - related stretching and bending modes . (ii) the fe h - related bending mode positions span from 740 to 790 cm for the examined iron hydride complexes, including those whose spectra are not shown here . Dft was also used to make a prediction about the possible position of this critical vibrational peak . However, all of the earlier dft calculations as well as normal mode analyses (before the ni fe was finally observed experimentally) pointed to a region with energies higher than 760 cm; some of them suggested a region higher (or much higher) than 800 cm and we experimentally searched up to 125 mev (or 1000 cm), but did not find anything . Fe regions was searched one to two times but led to no success at all . Although 620720 cm became the only region not searched with a great investment of beam time, the idea to search in this region was not at all popular at the time, because all the model complexes and theoretical calculations pointed to a much higher energy position . R model complex nihfe has its ni h fe at 758 cm, 140 cm higher than its highest - energy fe co peak at 617 cm, while ni then the question arose: should we perform a thorough search in this unlikely but unsearched region? Fe at 675 cm . As searching in the 620720 cm region was still not a mainstream opinion at that time, we first performed a balance measurement between acquiring a perfect fe co spectrum and searching for ni fe with a 4.5 mm nir - h sample at (improved) bl09xu . Therefore an even scanning time of 10 s / p was used between 400 and 740 cm instead of spending more time (e.g. 2030 s) in the ni (with a total scanning time of 680 s / p) a weak but positive peak at 675 cm was observed for the first time as shown in fig . 6(a), with the raw nrvs spectrum at the top and the converted pvdos at the center (10) and the bottom (1). We noticed that the 675 cm energy position is much lower than any of the previous dft or normal mode analyses predicted theoretical energy positions . It is actually still at least 32 cm lower than the lowest current dft value, which is at 707 cm [model vi in fig . 1(b 6)] (ogata, kramer et al ., 2015). The observed cts / s for various fe s, fe co and ni h fe in ni r are documented in fig . 4 [column 6 (not counting the columns showing the first argument was that the observed peak at 675 cm was still too weak . The second argument attributed this peak to a fe co - related feature, rather than to a fe h - related peak . However, there is no such fe co sub - feature in the mimic complex nihfe (ogata, kramer et al . These nrvs measurements on nir - d took 106 scans in total (= 40 + 30 + 36) and 2780 total s / p [= (40 20) + (30 30) + (36 30)], but concluded no peak in the same region . This suggests that the peak at 675 cm is related to a fe h vibrational mode . In order to resolve the first issue (to repeat the observation and to improve the spectral s / n) and to further clarify the second issue, two samples of nir - h and one sample of nir - d were re - measured at bl19lxu, which has a higher beam intensity and more nrvs cts / s than bl09xu . A similar pair of nrvs and pvdos for nir - h (blue, 24 scans, 480 s / p) and for nir - d (red, 20 scans, 400 s / p) are compared in fig . 6(b), illustrating much clearer evidence for the existence and the fe h - related nature for the 675 cm peak . From fig . 4 (column 6 for one measurement at bl19 and column 7 for one measuremnt at bl19, not counting the columns with the arrow symbols), the observed weak ni fe wagging mode is 2729 parts per million in intensity in comparison with the nuclear resonance peak, or 0.8% in comparison with the fe co (s), this ratio is about 14.816.6%, or, equivalently, ni h fe: fe co 1:61:7 . For such a weak signal, the apd s dark - current noise (instrument error) defines the best possible s / n because the instrumental error bar cannot be averaged lower with more signal statistics . Therefore a low (0.03 cts / s) dark current is the most important foundation for a successful observation of weak ni fe for a hypothetical 4 mm nir - h but measured at bl19lxu, which has 2.6 times more cts / s, is listed in column 5 of fig . The real signal level for a 4.1 mm nir - h at bl19 is listed in column 7: ni h fe = 0.12 cts / s for measurement 1 [and 0.13 cts / s for measurement 2 (not listed)]. In comparison with the hypothetical case (column 5), the real measurement (column 7) of ni h fe has a slightly lower cts / s level although the corresponding fe co has a higher cts / s level . This is because ni h fe: fe co is about 1:6 for nir - h (fig . Nir - h was measured once at bl09xu (680 s) and twice at bl19lxu (480 + 180 s). The signal level at bl09xu is estimated at (94 60)/680 = 0.05 cts / s, leading to a s / n = 0.05/0.03 1.7 and a not - so - clear nrvs (fig . 6b) is (139 81)/480 = 0.12 cts / s, corresponding to a s / n = 0.12/0.03 = 4, a much clearer observation for the ni the signal level for measurement 2 at bl19lxu is about 0.13 and s / n = 4.3 . The total equivalent bl09xu s / p (as defined in the experimental section) for nir - h in the nifeh region is 2396 s and the averaged signal level is 0.1 cts / s . Assuming as - high - as - possible statistics, this leads to s / n = 3.3 . As the real statistics are not infinite, the real s / n is slightly lower than 3.3 . Nir - d was measured three times at bl09xu and once at bl19lxu, equivalent to 3980 bl09xu s / p in the same region (versus 2396 s for nir - h). As there is no peak, there is no meaning to estimating the signal level or s / n for nir - d . However, a more equivalent s / p was used for nir - d because more time should be used to conclude that there is no signal than to find a weak signal . 7 summarizes three pvdos for nir - h and four pvdos for nir - d in the region around 675 cm . Again, all nir - h pvdos show a clear peak around 675 cm while none of those for nir - d show a peak in the same region, providing solid evidence that the observed 675 cm peak is real and is related to fe there are several experimental conditions which make this work successful and a super - concentrated (4 mm) and almost pure ni r) is one of the most important conditions but is also difficult to realise . We proceed with exchange ph of the buffer (7.4 5.0) and with a longer period (more than 8 h) of h2/d2 reduction . Monitoring samples integrity can never be more important than under these circumstances . The following three steps were used to monitor the samples integrity: (i) nir - h / d were first checked with ftir at the sample preparation laboratory before sending to spring-8 for nrvs measurements, as shown in fig . These subforms are distinguishable by ftir and differ in their protonation states but they are all ni r . R1 (co = 1946 cm), 16% nic (at 1962 cm) and a trace amount of ni r2 (co = 1933 cm). (ii) nir - h / d after nrvs measurements were also collected and checked with ftir and both ir spectra [blue for nir - h and red for nir - d in fig . 8(b)] show little change in comparison with those measured before sending for nrvs experiments (fig . 8a), confirming the integrity of the samples for the whole nrvs measurement . (iii) immediately prior to a nrvs measurement, in situ nrvs measurement in a sample s fe s region was another way of carrying out a last - minute check, as shown in fig . 9 . For example, the light blue curve shows one example of many good ni 7), while the black curve shows one bad (unexpectedly oxidized) ni the green and red spectra are the established nrvs for the oxidized and reduced dvmf h2ase (kamali et al ., 2013). R, the real control over the samples integrity is via the ftir measurements prior to and following the nrvs experiments, as shown in fig . 8 . This in situ examination was used only as a handy check that the sample was not oxidized (e.g. Fig . 9, black curve) before investing a long beam time on the sample . There are a lot of features in the fe s region which can be used to characterize the oxidation states of nife h2ase, including the peaks between 340 and 460 cm (kamali et al . . 4, column 4), a hypothetical 4 mm dvmf h2ase sample will have 36 cts / s in its fe s region, and three regular 5 s / p scans will produce 4590 total cts or a statistical s / n of 6.79.5 . As the dark current is much smaller in comparison with the fe s signal cts / s, the statistical s / n is close to the real s / n . Then a s / n of 79 should be sufficient to resolve the samples oxidation states . At bl19lxu, in principle, one scan is enough, but we usually took two 3 s / p scans in order to check the repeatability . We noticed that the low dark cts / s of the apd array (i.e. 0.03 cts / s) stays the same at bl19lxu, which has a higher beam intensity than bl09xu . If this dark - current level increases at bl19lxu, the increased cts / s will become less meaningful . We also noticed that the higher beam intensity at bl19lxu does not lead to higher sample temperatures (wang et al . This again illustrates the conclusion in our previous publication, which suggested that the hike in the samples temperatures in comparison with the sample base is mainly due to thermal radiation from the room - temperature window near the samples rather than from the x - ray irradiation (wang et al ., 2012). As the hrm is inside the same hutch as the nrvs measurement chamber, entering the hutch can disturb the hutch temperature and the performance of the hrm . A sample change or other regular operation, which lasts 30 min, will lead to a system recovery time of about 2 h. in the case of an emergency entry for 1 min, the system can be recovered in about 2030 min . As mentioned earlier, the energy scale can be calibrated either normally or with a quick - switch calibration procedure (wang et al ., 2013). For concentrated nir - h / d samples, the fe s peak at 365 cm (fig . 9) can also be used as a calibration reference because of its high signal level . The fe h or fe d stretching modes will have no overlaps with any possible nrvs peaks in the same regions and will allow a straightforward experimental identification of the hydride . However, in reference to the model complexes and dft calculations (ogata, kramer et al ., 2015), fe d has 1/5 of the intensity of ni h fe (see fig . 5) while fe h has 1/15 of that of ni h fe (not shown in fig . 5), presenting further challenges . Under the current experimental conditions, all these peaks are still not observable unless significant improvements in beam intensity, sample concentration and/or measurement solid angles are made . Different improvements and tests are underway . From the nidfe complex, the 711 cm ni d stretching bending peak (mixed with fe) has about the same intensity as ni h fe . However, it could be mixed with fe co and is not observable in ni this publication has presented the strenuous experimental journey to discover the first spectroscopic evidence for the existence of a bridging hydride in a [nife] h2ase s ni fe wagging mode has 2729 parts per million in intensity in comparison with the nuclear resonance peak, or 0.8% in comparison with a fe s feature . Improved sample concentration, increased photon flux and ultra - low noise level in the apd detectors all set the foundation for a possible nrvs measurement of the weak ni the decision and insistence on searching in the then unlikely energy region of 620720 cm was the key step towards successful observation of the ni the existence / non - existence of the 675 cm peak for nir - h / nir - d identified the peak as a fe h - related feature experimentally . Fe feature at 675 cm provided a critical observed reference for dft calculations and thus served as a key in the dft determination of the ni it will be of interest to other scientists who use synchrotron radiation for measuring dilute samples and/or weak spectroscopic features in general.
Thousands of children and adolescents across the united states suffer from musculoskeletal conditions each year [1, 2]. Common musculoskeletal conditions in children include cerebral palsy (cp), congenital muscular torticollis (cmt), duchenne muscular dystrophy, idiopathic clubfoot, idiopathic toe walking (itw), legg - calv - perthes disease (lcpd), limb length discrepancy, and neonatal brachial plexus palsy (nbpp). Musculoskeletal abnormalities and deformities can deprive children of physical activities, childhood experiences, and a healthy lifestyle . Besides the physical and psychosocial burden these conditions and injuries place on the child and family, these conditions also incur a financial burden for the patient and the healthcare system as multiple hospital visits are often required . Proper and timely treatment including standard approaches such as physiotherapy, casting, bracing, and surgery is essential to ensure the child optimal growth and development . Besides these traditional modalities, the use of botulinum toxins appears as a promising treatment on its own, as an adjunct to other treatment modalities and as an alternative to surgery . Several authors have suggested the use of btx - a in children with musculoskeletal conditions, yet the evidence supporting its safety and effectiveness is not well established . The use of btx - a in children with cp has been widely documented [35] and is beyond the scope of this paper . The objectives of this systematic review were to establish the evidence on the effectiveness, safety, and functional outcome of btx - a in children with musculoskeletal conditions . Botulinum toxin is an extremely potent, naturally occurring poison resulting from the fermentation of the anaerobic spore - forming bacterium clostridium botulinum . These toxins cause flaccid paralysis by blocking acetylcholine release, which is required for muscle contraction at the neuromuscular junction . Thus, the toxins have the capacity to reduce muscular activity in a dose - dependent manner . Muscle weakness occurs within a few days to one week after local injection, peaks within two weeks for several weeks, and then plateaus in milder form (the desired clinical effect) before gradually returning to baseline . Recovery from the toxin - induced paralysis begins with resprouting of axon terminals and slow recovery of the neurons ability to release acetylcholine, which results in nerve conduction to be reestablished . Clinical observations suggest that these neurotoxins have three mechanisms of action: paralytic, antisecretory, and analgesic (antinociceptive). A number of studies suggest that several pathways play a role in the analgesic effects of botulinum toxins, such as in conditions of pathologic muscle overactivity (dystonia and spasticity) and in the role of the calcitonin gene - related peptide in the afferent signaling of pain . The fda has approved a total - body dose of 400 units administered every 12 to 16 weeks or at longer intervals to avoid toxicity . Identifying the appropriate muscle sites for injection is done through palpation, electrical stimulation, electromyography, ultrasound, fluoroscopy, and computerized tomography depending on the muscle size and location . A needle between 23 and 27 gauge is selected based on muscle depth, difficulty palpating the muscle, and use of electromyography . There are seven different serotypes of the neurotoxin, named botulinum toxin (btx) types a to g. although they all inhibit acetylcholine release from nerve terminals, they differ according to their intracellular protein targets, potency, dosing, and duration of effect . Btx - a is the serotype that has been most studied in terms of therapeutic application . Btx - b and btx - f have also been used in clinical practice, but are less potent than btx - a, and have a shorter duration of action . Btx - b has also been shown to have regional and systemic anticholinergic adverse side effects, which limits its clinical use . There are currently three types a and one type b brand of botulinum toxins available in the us market . In 2010, the us food and drug administration (fda) announced generic names for all of the versions of injectable botulinum toxins . This change in terminology is expected to differentiate between these different brands and provide each brand with its own identity thereby improving its clinical use and reducing errors and misinterpretation . Hence, onabotulinumtoxina (botox by allergan inc in the united states), abobotulinumtoxina (dysport by ipsen in france), incobotulinumtoxina (xeomin by merz pharmaceuticals gmbh in germany), and rimabotulinumtoxinb (myobloc / neurobloc by solstice neurosciences in the united states) are the new four generic names used in the usa . Btx - a was first approved by the fda in 1989 for the treatment of strabismus and blepharospasm (two eye muscle disorders), making it the first botulinum toxin type a product approved in the world . In the usa, btx - a is also approved to treat cervical dystonia and severe primary axillary hyperhidrosis in adults . Recently, the fda has also approved the use of btx - a to treat increased muscle stiffness in the elbow, wrist, and finger muscles in adults with upper limb spasticity . In addition to its therapeutic uses, the same formulation of btx - a was approved by the fda in 2002 under the trade name botox cosmetic to improve the look of facial wrinkles in adults less than 65 years of age . Although the use of btx - a has not yet been approved for use in children, it has been used in a variety of clinical conditions both for its neuromuscular and analgesic effects due to its safe, predictable, and reversible effects on motor weakness . These off - label indications include cmt, cp, idiopathic clubfoot, itw, lcpd, lower limb lengthening and nbpp . Btx - a is used in these children to decrease spasticity, manage postoperative pain, and improve quality of life . The most common use of btx - a in children is for the treatment of spasticity in cp . Studies have shown that btx - a decreases muscle tone in children with upper and lower limbs spasticity associated with cp and can help prevent the development of muscle contractures and bony deformities, as well as improve upper limb movement and function [1517]. Patient selection, btx - a dosing, dilution and administration, identification of muscle groups, and outcome measurement must be carefully considered [13, 16]. Adverse events following botulinum toxin injection have been found to be mild, temporary, as well as dose and site related . These may include local reactions, such as bruising and pain at the site of injection, excessive localized muscle weakness, and incontinence . Systemic effects are very rare and may include flu - like symptoms, headaches, light - headedness, fever, chills, hypertension, diarrhea and abdominal pain, generalized weakness, dysphagia, dry mouth, and subsequent aspiration . These are far less common, are generally short - lived, and may result from the systemic spread of the toxin to adjacent muscles . A boxed warning label describing the spread of the toxin and its potentially life - threatening effects appears on the labels following the death of a child following btx - a injections . However, until today, no causal relationship confirmed the evidence relating this specific adverse event to the toxins . Caution should be exerted when injecting btx - a in children with preexisting swallowing or respiratory problems . The incidence of antibody - mediated resistance in long - term treated patients ranges from 3 to 23%, depending on the patient sample, treatment regimen and toxin preparation . A recent australian study prospectively documented the presence of adverse events in 334 children with cp in the month before and in the month after btx - a injection . They found that the children had significant morbidities prior to the injection, adverse events were present in 23.2% of children, and no deaths occurred . Cmt is common and refers to unilateral contracture of the sternocleidomastoid muscle that restricts the infant's range of motion at the neck . Infants with cmt display head tilt toward the shortened side, which is often combined with rotation of the head to the opposite side [20, 21]. It is reported to occur in 1 infant in every 250300 live births [21, 22]. Manual stretching is still the most common form of treatment, and about 90% of cmt resolves with stretching exercises . When conservative treatment is ineffective, surgery is considered . However, as an alternative to invasive surgical intervention, btx - a may be an option to increase the effectiveness of stretching on the side of the contracture and allow strengthening of overstretched and weakened muscles on the opposite side of the neck [12, 21, 22]. It is a progressive x - linked recessive disorder and is caused by a defective gene for dystrophin affecting approximately 1 out of every 3,600 male infants . The proximal muscles of the lower extremities are affected first, with decreased range of motion, flexion contracture of the hip and knee, and extension contracture of the ankle . Symptoms usually appear before age 6 and typically include fatigue, muscle weakness, and progressive difficulty in walking . Braces may improve mobility and self - care function, and a wheelchair is often used by age 12 . There is no known cure for duchenne muscular dystrophy and by the late teens or twenties the condition is severe enough to shorten life expectancy . Treatment aims to control symptoms to maximize quality of life and maintain muscle strength and function . Btx - a may be indicated to decrease muscle contractures of the lower extremities and facilitate standing and mobility . In this congenital deformity, clubfoot is one of the most common birth defects, occurring in 13 per 1000 live births . A child with an untreated clubfoot will walk on the outer edge of the foot instead of the sole, develop painful callosities, be unable to wear shoes, and have painful feet that often limit activity . Nonsurgical modalities include serial manipulation and casting, such as ponseti's technique [24, 25], as well as taping, physical therapy, and continuous passive motion . Surgery is indicated if satisfactory clinical and radiographic correction by nonsurgical methods is not obtained . Btx - a injection into the child's calf muscle is indicated to facilitate nonsurgical techniques, to supplement surgical release and to serve as an alternative to achilles tendon lengthening by reducing the tone in the most contracted muscles [12, 14]. This condition is present in children older than 3 years of age still walking on their toes without any neurological, orthopaedic, or psychiatric diseases . Treatment recommendations include physical therapy, casting, bracing, surgical release, and achilles tendon lengthening . Btx - a may be useful to manage the contractures in cases in which toe walking recurs despite conservative and surgical treatment . Lcpd is a degenerative disease of the hip joint affecting about 10.8 children of 100,000 children and is more common in boys . It is characterized by an interruption of the blood supply of the head of the femur with loss of bone mass and joint deformity at the hip . The disease is typically found in young children, and it can lead to osteoarthritis in adults . Common symptoms include hip, knee, or groin pain, exacerbated by hip / leg movement, reduced range of motion at the hip and painful and limping gait . Physical activity such as standing, walking, running, and kneeling may cause severe irritation or inflammation of the damaged area . The goal of treatment is to prevent deformity of the femoral head and avoid severe degenerative arthritis later on . Btx - a may be indicated to weaken selected muscles to restore muscle balance at the hip joint . Children undergoing lower limb lengthening using an external fixator exhibit excellent results in most cases, yet the postoperative pain can be significant and often requires prolonged use of analgesics and even narcotics [28, 29]. Other aspects of the limb lengthening, such as the osteotomy and incision sites, gradual distraction increasing soft tissue tension, range of motion exercises, and gait training result in ongoing pain for weeks after the application . Appropriate pain management is essential for optimal quality of life and functional outcomes in children undergoing limb lengthening . Btx - a has been shown to reduce postoperative pain in children secondary to reduced muscle spasm and may be indicated in children undergoing lower limb lengthening to alleviate spasm and pain during the lengthening process . Nbpp is defined as a flaccid paresis of the upper extremity secondary to unwanted muscular cocontraction or inappropriate activation of antagonist muscles due to increased forces of distraction to the neck during delivery . Associated injuries may include fractures to the clavicle and humerus, facial nerve palsy, and torticollis . Incidence varies between 0.38 and 3 per 1000 live births in industrialized countries and occurs more frequently in infants born over 4 kg, breech deliveries, maternal diabetes, and vacuum / forceps extraction . While early physical therapy yields complete return to function in many infants, infants whose elbow flexion and shoulder abduction have not recovered before 6 months are indicated for surgical reconstruction of the brachial plexus . A novel approach for children with nbpp injuries is the therapeutic use of btx - a to inhibit unwanted cocontractions and activate antagonist muscles . Early treatment of these children with btx - a injections may also result in stronger, normal muscles and may prevent the development of glenoid dysplasia [12, 31]. A literature search using the electronic databases medline, pubmed, cochrane, trip, and web of science for published articles in english from 1980 to september 2011 was conducted using botulinum toxin, congenital muscular torticollis, duchenne muscular dystrophy, idiopathic clubfoot, idiopathic toe walking, legg - calv - perthes disease, lower limb lengthening, neonatal brachial plexus palsy, and relevant search terms . To be included, a study had to be written in english and to include children between 0 to 21 years of age with one of the above - mentioned musculoskeletal conditions . Research studies that were included in this review were classified as levels i to iv, based on the american academy of neurology (aan) levels of evidence . Data was independently extracted by two reviewers (n. dahan - oliel and b. kasaai). The majority of studies were on children with nbpp (n = 10), followed by idiopathic clubfoot (n = 5), cmt (n = 4), itw (n = 3), duchenne muscular dystrophy (n = 1), and lower limb lengthening (n = 1). No studies that looked at the effect of btx - a in children with lcpd were found . Two of these studies [32, 33] included both children and adults but were included as separate results for children were provided . In total, outcome was provided for forty - five children aged 3 months to 18 years following btx - a injections . The following muscles were injected in isolation or in combination: sternocleidomastoid, trapezius, splenius, and scalenus . Two studies reported mild dysphagia, neck weakness, neck bruising and soreness, and brief fever following injection in five children [34, 35]. There were variations in btx - a dosage, number of injections per muscle, length of follow - up, and outcomes assessed between studies as well as within the same study, which limited comparability of findings . It is difficult to attribute clinical improvements to the btx - a treatment since the children in these studies often received a combination of both btx - a injections and physical therapy . Furthermore, none of these studies compared btx - a treatment with either traditional treatment or surgical treatment . One study on 15 infants who had a significant risk of progressing to surgery because of severe torticollis found promising results . Indeed, only the oldest infant required surgical release following btx - a injections, while the other 14 infants showed significantly improved neck rom and did not require surgery . These findings suggest that btx - a may be a safe and effective treatment modality when traditional treatments (home program, physical therapy) do not yield acceptable results . Btx - a treatment may obviate surgical interventions in certain cases, but randomized controlled trials with larger samples are needed to confirm this finding . Although it seems that the initial injections of btx - a should be administered at a young age, the exact age is not yet established . An 11-year - old boy was injected with btx - a for tightness in the left knee flexors in order to enable standing exercises . Knee range of motion was increased by 20 degrees following injection, with no side - effects . Btx - a may be indicated in children with duchenne muscular dystrophy when temporary improvement of range of motion is needed to minimize knee contracture and encourage exercises for muscle stretching, prevention of osteoporosis, and retaining lung function . The first study by delgado and colleagues included four patients who had severe clubfoot deformities and rapidly reached a plateau following physical therapy . Btx - a injections helped most of the patients, despite their severe deformity, allowing two of them to avoid surgery . The remaining two patients had a demyelinating neuropathy and did not respond to btx - a treatment . Another study by mitchell and colleagues reported on three children under the age of 13 months who had a recurrence of their deformity after surgery and found a marked improvement of the deformity following btx - a injection and application of molded plaster casts . The largest study by alvarez and colleagues [37, 38] included 51 children . Following ponseti - type manipulations, casting, and btx - a injection, all but one child improved in terms of dorsiflexion and a decrease in the severity of the deformity was noted . Bracing was provided to maintain the correction . At the five - year follow - up visit, 48% (31 of 65 clubfeet) successfully responded to a single btx - a injection and experienced no recurrence over the follow - up period . At least one repeat btx - a injection was required in 34 clubfeet, and surgery was required in 10 clubfeet . These four studies found a positive effect of btx - a as an adjunct to manipulation, casting, and physical therapy to correct muscle imbalance and to correct recurrent deformity in idiopathic clubfoot . All five studies concluded that btx - a may be an effective and safe treatment alternative and can decrease the number of patients requiring surgery . A limitation of these four studies is that they did not include a control group and that the results cannot be attributed solely to btx - a as other treatment modalities were used . Cummings and colleagues conducted a randomized double - blind controlled trial on 20 infants (32 clubfeet) comparing a single btx - a injection to placebo following serial manipulation and casting according to the ponseti technique . The study found no significant difference in time of correction, need for tenotomy, or relapse between both groups . However, this trial included a small sample size and low btx - a dosage that may have compromised positive findings of btx - a . No adverse events were reported following btx - a injections in children with clubfoot . In order to address the need for additional btx - a injections, a larger trial is required with regular follow - up as well as a good maintenance - bracing program, so as to provide each child with an individualized treatment . The exact dose and number of injections three studies [4244] reported improvement in gait pattern, function, and decreased toe walking severity following btx - a injection to gastrocnemius and soleus muscles . In addition to the btx - a injection, children also received a home exercise program, physical therapy, and orthotics . Further studies are needed to evaluate whether repeated injections and btx treatment in combination with other treatment interventions (such as orthotics and physical therapy) improve outcome . This was a pilot randomized controlled trial comparing the effects of btx - a injection versus placebo at the time of surgery . Fifty - two children with limb length discrepancies of various etiologies, as well as children with surgical correction of clubfoot deformities, were included . Findings showed that compared to placebo, the btx - a group had a trend for lower pain at middistraction, less parenteral pain medication after - surgery, higher functional mobility scores, and better quality of life at three of five time points, although these differences were not statistically significant . No adverse events related to the btx - a injection were reported, indicating that btx - a may be safe and effective in alleviating pain, improving functional mobility and quality of life in children undergoing lower limb lengthening and/or deformity correction . Future studies with larger sample sizes are required and with homogeneous study populations to verify whether btx - a injections are beneficial at the time of surgery in these children . Ten studies [4655] reported on the outcomes of using btx - a, in which one study by hierner and colleagues was a follow - up report of the previous study by rollnik and colleagues . Although all children were diagnosed with nbpp, there were a number of variations between studies in the specific mechanism of injury and in the underlying limitations, such as in severity of biceps - triceps cocontractions, persistence in shoulder paralysis, medial rotation deformity of the shoulder, response to serial cast treatment, and posterior shoulder subluxation or dislocation . However, different muscle injection sites were used across the studies, indicating an individualized protocol according to each child's condition and needs . Four studies reported administering additional btx - a injections, as needed . All ten studies reported positive outcomes following btx - a injections, including improved ability to make hand - to - mouth movements, avoidance of open surgical procedures, improved active shoulder abduction and elbow extension, better functional scores, and reduction of triceps cocontractions during elbow flexion . Two studies [46, 48] reported that those children who did not experience improved outcomes following btx - a injections were older . Desiato and risina found that the gain in shoulder abduction was directly related to younger age (r = 0.6). Transient weakness was reported in a 6-year - old female and mild to moderate discomfort at the injection site was reported in two children . These findings indicate that btx - a may be a promising treatment modality in young children with nbpp; however, stronger methodologies are required to confirm the effectiveness of btx - a for this condition . The objectives of this systematic review were to establish the evidence on the effectiveness of btx - a in several musculoskeletal conditions in children and to show whether these studies reported improved functional outcome . Out of the 24 studies reviewed, only two randomized clinical trials were conducted, one in children with clubfeet and the other in children undergoing lower limb lengthening . Furthermore, treatment of children with musculoskeletal conditions is often multimodal, including bracing, casting, rehabilitation, and home programs . Therefore, attributing the improvements in outcome to a single intervention, such as btx - a, is not straightforward . A recent systematic review on the indications for the use of btx - a treatment for children with nbpp was conducted by gobets and colleagues . They included 10 full - text papers and six congress abstracts, involving 343 children . Four groups of indications were identified: internal rotation / adduction contracture of the shoulder, limited active elbow flexion, limited active elbow extension, and pronation contracture of the lower arm . Overall, positive results were reported for all except the indication for limited active elbow extension . However, only one study was comparative in nature; all others were classified as having a low level of evidence . These authors conclude that multicentre randomized controlled trials are needed to provide better evidence of btx - a in this population . These factors include the different dosages and commercial sources of btx - a used across the different studies . Two brands of btx - a were used across the 24 studies (allergan and dysport). Btx - a brand was not always specified in the reviewed studies and this information was made available in several cases by contacting the authors . These two preparations should not be used interchangeably, either in terms of predicting outcome or in determining doses to be used . Though the units are not interchangeable, various published reports support a conversion ratio from 1: 5 to 1: 3 . The technique used to identify which muscle groups will be injected also varied (palpation technique, electrical stimulation, etc . ). Indeed, different studies have used different techniques and thus may lead to varying outcomes following btx - a injection . There is no consensus as to the exact age at which a child with a musculoskeletal condition should be first administered btx - a . Age of injection varied across the different studies reviewed . However, several authors [46, 48, 54] found that the younger children at the onset of btx - a treatment for nbpp benefited most compared to older children . Another factor to keep in mind is the need for administering repeat btx - a injections for sustained benefit . Some authors administered repeated btx - a doses, whereas other authors gave just one dose, even in cases when the children did not demonstrate improved outcome following the initial btx - a injection . This may have been due to financial and time restrictions as repeated injections are both expensive and time - consuming . Findings indicate that repeat btx - a doses appear safe in children with musculoskeletal conditions . Longer follow - up periods are required to inform health care providers whether the benefits of btx - a are temporary and transient and, then if indicated, at which time should an additional injection be administered . They reported that 48% of clubfeet were successfully corrected after a single dose of btx - a in 44 children . Serious adverse events related to btx - a injection were not reported in the studies included in this review . Minor adverse events such as transient muscular weakness and local discomfort at the injection site were reported in few studies . While this may be suggestive of the clinical safety of btx - a, it is important to note that several studies did not actually report on the presence or absence of adverse events . Therefore, more rigor is required in reporting these events to establish the safety of btx - a in children with musculoskeletal conditions . Btx - a is a promising treatment adjunct in improving functional outcomes in children with musculoskeletal conditions by causing a flaccid paralysis of the affected muscles . Further studies should include a prospective methodology, longer follow - up periods, and comparison group and evaluate whether repeated injections are required to improve the outcome of children, thus providing evidence on the effectiveness and safety of this drug in children with musculoskeletal conditions . N. dahan - oliel and b. kasaai carried out the systematic review and data extraction.
Improving quality of life and quality and management of care of clients with a schizophrenic disorder by the use of a comprehensive internet based disease management (dm) platform . Presenting factors influencing the implementation of an internet based dm platform for clients with schizophrenia . Two mental health care institutions in the south of the netherlands participate in the implementation of the digital instrument (the dm platform). The implementation of the dm platform will be followed by a process and an effect evaluation . Measurement instruments: in the effect evaluation, quality of life, number of symptoms of schizophrenia, quality of care, social functioning, self management, and insight in schizophrenia and its treatment will be measured . Process evaluation: semi - structured interviews will be held with different (formal primary and secondary, and informal) caregivers, managers and clients of each institute . These interviews will be conducted before the implementation of the platform and one, two and three years later . In the digital platform the guideline for people with schizophrenia will be implemented and also information for clients and their home caregivers . When the platform is ready clients will be able to communicate digital with their caregivers in the mental health institute and with their home caregivers and gps . The effect evaluation starts in september 2009, the process evaluation has started in may 2009 . We will have the first results in june 2009; they will be used to adjust the implementation and the development of the dm platform . In the meantime we can report about the building of the dm platform and what the expectations from the future users are . What are the factors that influence the process of implementation of a digital tool that provides help and care on a distance? In what way does the digital tool (care from a distance) stimulate people who need chronically care to be more self - supporting? In what way does the digital tool support professionals to be able to stimulate people with schizophrenia to be more self - supporting?
Psoriasis is a common, chronic, disfiguring, relapsing, inflammatory, and proliferative condition of the skin . Long - term treatment of moderate - to - severe psoriasis is challenging and often requires a combination, rotational or sequential therapy, with two or more systemic agents . Although it had been avoided earlier, combination therapy with methotrexate and cyclosporine is being increasingly found to be safe and useful in the management of psoriasis . We report here the case of a 57-year - old patient with unstable psoriasis, who developed unexpected methotrexate toxicity and cyclosporine - induced thrombocytosis in a span of two weeks of being given both drugs sequentially . We present this case to highlight the as - yet - unknown toxicities and interactions encountered with the administration of methotrexate and cyclosporine in the treatment of psoriasis . A 57-year - old male, who was known to be suffering from psoriasis vulgaris for the past 10 years, presented to us with features of unstable psoriasis for a duration of two weeks . He had been previously treated for multiple exacerbations with methotrexate (cumulative dose - 2285 mg), cyclosporine, and topical steroids, on separate instances . He was a known diabetic for the past 10 years, on regular treatment with tablet metformin 500 mg twice a day . His baseline complete blood count showed leukocytosis (leucocyte count - 18,300 cells / mm; platelet count- 5 10/mm) and the liver function tests were within normal limits . In view of the suspicion of unstable psoriasis, inj . After 24 hours, he developed multiple pustules over the nape of the neck and multiple oral erosions, resulting in severe odynophagia, with the existing lesions becoming more erythematous [figure 1]. The hemogram showed a fall in the leukocyte counts (7,700 cells / mm). Improvement in his blood count was also noted, as they returned to normal in four days (leucocyte count - 9,700 cells / mm; platelet count - 4.11 10/mm). The causality assessment, done using the naranjo algorithm, revealed a probable adverse drug reaction . Mild erythema and hyperpigmentation of plaques three days after starting methotrexate and folinic acid following this, the patient was started on cyclosporine 100 mg, twice a day, to control the unstable psoriasis . One week after administration of cyclosporine, it was noticed that the platelet count increased three times from the baseline values (9.0 10/mm) when tested, on multiple occasions . The platelet count dropped to a high normal value (5 10/mm) four days after withdrawing the drug, thus confirming our suspicion . The naranjo scale scoring was 7, which again implied a probable adverse drug reaction . The patient was then started on acitretin 50 mg once a day and was discharged after resolution of the lesions [figure 2]. Methotrexate and cyclosporine are considered to be the foremost treatment options of moderate - to - severe psoriasis . As both carry possible side effects and complications, factors predictive of methotrexate toxicity in patients with rheumatoid arthritis include absence of folate supplementation, high body mass index, prior gastrointestinal events, lower age group, female sex, renal impairment, change in dose, drug interactions, and infection . It remains unexplained as to why this patient, who had received methotrexate for many years, suddenly developed methotrexate toxicity after a relatively low dose . He was given a low dose of parenteral methotrexate, as he developed unstable psoriasis while on a low dose of oral methotrexate . The role of change of route of administration in causing methotrexate toxicity remains to be studied . An alternative explanation for thrombocytosis in our patient could be a reactive thrombocytosis set off by methotrexate toxicity or by the disease itself . However, the normal platelet count after withdrawal of cyclosporine is more suggestive of the former . Reactive thrombocytosis is the exaggerated physiological response to a primary stimulus and is also known to accompany chronic inflammatory diseases like psoriasis . Yasumoto et al . Also reported reactive thrombocytosis in two patients with psoriasis, with associated raised il-6 (interleukin-6) levels, thus supporting the theory that thrombocytosis could be caused by the disease per se . However, the fact that il-6 also has a role in the pathogenesis of psoriasis as t memory / effector cells that are chronically activated and poorly suppressed by regulatory t cells must be remembered . Il-6 signals through stat3 and allows the escape of t memory / effector cells from t regulator - mediated suppression in a murine system . Cyclosporine can induce il-1 expression in circulatory leukocytes and this may be sufficient to induce il-6 production in some tissues . This is a plausible explanation to the increase in platelet counts during cyclosporine therapy in our patient . The role of platelets in the inflammatory process is increasingly being recognized, in addition to their function in hemostasis and thrombosis . Hence, the effect of the inflammatory process of unstable psoriasis on the platelet counts or vice versa is also an interesting postulate in our case; the temporal course of events do not, however, suggest a correlation . We found it curious that our patient developed cyclosporine - induced - thrombocytosis a week after he recovered from unexplained methotrexate toxicity . There are no reports on similar hematological manifestations because of the potential interactions between methotrexate and cyclosporine when administered concurrently or sequentially . However, a study on the cytogenetic effect of methotrexate on human cells in vivo showed that methotrexate had a chromosome - breaking effect on human bone marrow cells . Recent studies have highlighted that methotrexate and cyclosporine can be co - administered in patients with difficult - to - treat psoriasis . However, this case shows that there is still a need for caution, as late sequelae of long - term administration of the drugs are still unknown . We present this case to highlight that unknown long - term toxicities and unexplored interactions between systemic agents used for psoriasis still remain a chink in our therapeutic armamentarium for this common disease . This defect can be repaired by well - planned cohort studies and meticulous documentation of records of patients.
Fibrosarcoma of the breast is included in this group of sarcomas and this tumor is always malignant, although the malignancy may vary in degree . Primary breast fibrosarcoma is a rare neoplasm . In a review of the literature, barnes and pietruszka reviewed all the cases of breast sarcoma that were seen at the health center hospitals of the university of pittsburgh during the 1945 - 1976 period, and they found that only 10 cases of primary breast sarcoma had been reported . Further, adem et al . Reported that primary breast sarcoma comprised only 0.0006% of all breast malignancies presenting at his institution from 1940 to 1999 . However, the exact incidence of primary breast fibrosarcoma is not known nor has it been reported in the literature . Reports in the medical literature with the radiologic imaging of primary breast fibrosarcomas are also very rare . To the best of our knowledge, there has been no prior report of the sonographic imaging of primary fibrosarcoma of the breast in the english medical literature . Here we report on the mammographic and sonographic imaging and pathological findings of a low grade, primary breast fibrosarcoma . A 47-year - old woman presented with a painless palpable mass in her left breast . A physical examination revealed a 3 cm size, relatively well - defined mass in the upper outer quadrant of the left breast . The mammograms showed a 3 cm, ovoid, partially obscured, partially circumscribed, and high - density mass without internal calcifications in the upper outer quadrant of the left breast (figure 1a, b). A sonogram showed a 2.5 cm, ovoid, circumscribed and homogeneous hypoechoic mass with posterior acoustic enhancement at the 2 o'clock site of the left breast (figure 1c) and color doppler image showed vascularity in the peripheral portion of the mass (figure 1d). We therefore classified the mass as category 4b according to the breast imaging reporting and data system (bi - rads). We then performed an ultrasonographic - guided core needle biopsy, and the histopathological examination of the biopsy specimen revealed the presence of a cellular, spindle cell proliferative neoplasm with intermittent mitosis . These findings did not allow differentiation between spindle cell mesenchymal neoplasm and fibroepithelial neoplasm, and the findings could not provide a diagnosis . Therefore, surgical excision was performed, and the gross specimen showed a firm, fleshy, well circumscribed, round mass that was gray - white and it measured 3.5 cm at the greatest dimension . Histopathological examination showed a high cellular, spindle cell tumor that displayed an interdigitating fasciculated growth pattern, the so - called herringbone pattern (figure 2a). The tumor cells were fusiform or spindle shaped cells that varied little in size and shape, and they had scanty cytoplasm with indistinct cell borders . The mitotic activity was up to 9 - 10 per 10 hpf (figure 2b). The negative immunostaining results for s-100 protein, smooth muscle actin and many kinds of keratin, including ck ae1/ae3, high molecular weight ck, low molecular weight ck, ck7 and epithelial membrane antigen, distinguished this tumor from spindle cell malignant melanoma or malignant peripheral nerve sheath tumors, leiomyosarcoma and metaplastic carcinoma (figure 2c). She had no postoperative complications, and she remains well without signs of local recurrence or distant metastases 10 months following her surgery . Pure stromal sarcoma of the breast includes fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, malignant schwannoma, osteogenic sarcoma and chondrosarcoma . Adult fibrosarcoma is a malignant tumor that is composed of fibroblasts with variable degrees of collagen production and in classical cases a herringbone architecture, according to the world health organization . Fibrosarcomas can occur anywhere in the body, but they usually occur primarily in the extremities . . Reviewed only 25 cases of primary breast sarcoma that were diagnosed pathologically at a london hospital during an 80-year period . Among these the peak incidence of primary breast sarcoma occurs in the fifth and sixth decades of life . However, roberson has reviewed the literature and evaluated 49 reported patients with fibrosarcoma of the breast, and half of them were between the ages of 41 and 60 . These patients usually present with a complaint of a lump in one breast of two to six months duration and pain was present in over one - third of the cases . In contrast, the clinical presentation of a common primary breast sarcoma is a painless breast lump . The size of primary breast sarcomas is variable and it ranges from less than 1 cm to larger than 40 cm . The prognosis is usually based on the size of the tumor and the histological grade . The stromal sarcomas were classified as malignant fibrous histiocytomas (11 cases), fibrosarcomas (2 cases), leiomyosarcomas (2 cases) and liposarcoma (1 case). In that study, the metastasis - free survival rate was significantly correlated only with the histological grade, which consisted of the tumor differentiation, the presence of tumor necrosis and the mitotic activity . The clinical courses and survival of the cystosarcoma and stromal groups were identical, which raises questions about the clinical value of this pathologic distinction . All the local recurrence, metastasis or death occurred within 30 months, although the follow - up was much longer . Of the 55 patients, 17 had breast - conserving therapy and 38 women had mastectomy . The mean patient age at presentation was 52 years (range, 22 to 82 years). The types of sarcoma included angiosarcoma (18), malignant fibrous histiocytoma (11), stromal sarcoma (8), liposarcoma, leiomyosarcoma, dermatofibrosarcoma protuberans (4), osteosarcoma (3), fibrosarcoma (2) and rhabdomyosarcoma (1). Follow - up information was available for 53 patients with a mean follow - up of 81 months . Twenty - nine of the 53 patients (55%) developed recurrent sarcoma and 23 patients (43%) died of their disease . Twenty - seven patients had no evidence of recurrence, and 3 patients were alive with disease at the last follow - up . Twelve of 18 patients (67%) died of angiosarcoma, compared with 11 of the 32 patients (34%) with all the other types of sarcomas combined . Of the 34 patients who did not receive adjuvant chemotherapy or radiation, 13 died of their disease (38%), as compared with 10 of the 16 patients (63%) who did receive adjuvant therapy . In that study, elson et al . Reported the mammographic findings of 5 cases with fibrosarcoma of the breast . In their study, the mammograms showed high - density masses with margins varying from poorly defined to well - defined and the diameters ranged from 1.5 cm to 7.0 cm . Calcified osseous elements were present in 1 of the masses . However, these findings were nonspecific . In the case presented here, the mass appeared as an ovoid, partially obscured and partially circumscribed, high - density mass on mammography and this is also a nonspecific finding and it could not distinguish fibrosarcoma from other breast carcinomas . On ultrasonography, the mass appeared as an ovoid, circumscribed and homogeneous hypoechoic solid mass with posterior acoustic enhancement . In our patient, the size of the tumor was 3 cm and there was no lymph node or distant metastasis . The tumor stage was a 1a according to the american joint committee on cancer . According to the national comprehensive cancer network clinical practice guidelines in oncology, surgery is the primary treatment for low - grade stage 1 tumors and it is considered definitive if the tumor free margins are greater than 1 cm or the fascia plane is intact . For surveillance, stage i tumors are routinely followed with taking the recent medical history and conducting a physical examination every 3 - 6 months for 2 - 3 years and then annually . Chest imaging should also be considered every 6 to 12 months . Because these patients' risk never returns to zero, long - term follow - up is indicated and this should include mri or ct scanning . If the final tumor - free resection margins is 1 cm or less, then radiotherapy should be considered . In our case, local recurrence and distant metastasis did not occur in our patient 10 months after surgery . In conclusion, fibrosarcoma of the breast is a rare tumor and the imaging results make it difficult to differentiate this type of lesion from other malignant masses.
Helicobacter pylori (h. pylori) is a type of bacterium that frequently colonizes the lining of the stomach . H. pylori infection is recognized to be the most important acquired factor in the etiology of ulcers of the stomach and duodenum . H. pylori infection was also shown to be associated with active gastritis, gastric carcinoma and gastric mucosal associated lymphatic tissue (malt) lymphoma . It is believed that eradication therapy is a powerful way to cure h. pylori - related diseases . But the relationship between h. pylori and gastroesophageal reflux disease (gerd) is still unclear, and the effect of h. pylori eradication on gerd treatment is unknown . In this study, we selected patients with reflux esophagitis to explore the effect of h. pylori and its eradication on reflux esophagitis treatment . Patients from digestive disease center of peking university third hospital, wu han union hospital, shanghai changhai hospital, and the first affiliated hospital, sun yat - sen university were enrolled between january 2007 and october 2010 . Included criteria were: almost 1870 years of age, typical reflux symptoms (heartburn, acid regurgitation and chest pain), and diagnosis of reflux esophagitis (los angeles [la] a - d degree). The la classification was used for the patients with reflux esophagitis, and those with reflux esophagitis la - a to la - d were all enrolled . Patients were excluded with: peptic ulcer, carcinoma of upper gastrointestinal tract, esophageal varices, zollinger ellison syndrome, esophageal stricture, barrett's esophagus, other severe gastrointestinal disease (for example ulcerative colitis), and accompanying severe diseases of other systems . Pregnant or lactating females, and patients who had undergone proton pump inhibitor (ppi) or bismuth therapy during the preceding 2 weeks were also excluded . Biopsy specimens for rapid urease test (rut) were obtained from biopsy forceps with a sterile needle and assessed immediately; the results were examined by an experienced observer blinded to the clinical details . The biopsy specimens for pathology were examined by an experienced pathologist after warthin starry (ws) staining . A sample was considered h. pylori positive with both rut and ws staining results positive . H. pylori negative samples showed negative results in both tests . When discordant results were obtained for rut and ws, the patients were excluded from the study . Biopsy specimens were examined by ws staining, and samples were considered h. pylori positive with positive ws staining . Before and after treatment, patients underwent gastroscopy and were examined by experienced doctors . The severity of reflux symptoms was scored by the frequency and severity of the symptoms [table 1]. Scores of frequency and severity of reflux symptoms we divided a day into three periods: from breakfast to lunch, lunch to supper and supper to breakfast . If a reflux symptom appears in one, two or three periods, a score of one, two or three, was attributed, respectively . H. pylori positive patients were first randomly divided into eradicated and uneradicated groups by random envelope . In the eradication group, patients underwent h. pylori eradication for 10 days, then received esomeprazole 20 mg bid for the following 46 days . Two eradiation regimens were randomly selected in this study: the eac regimen consisted of esomeprazole 20 mg bid, amoxicillin 1.0 bid and clarimothin 0.5 bid; in the sequential regimen, esomeprazole 20 mg bid and amoxicillin 1.0 bid were administered for the first 5 days, while esomeprazole 20 mg bid, clarimothin 0.5 bid and tinidazole 0.5 bid were given for the following 5 days . In h. pylori positive uneradicated and h. pylori negative groups, patients were treated with esomeprazole 20 mg bid for 8 weeks . Afterward, all patients underwent gastroscopy again, and the ws staining test was repeated . Therefore, three groups were obtained: h. pylori positive eradicated, h. pylori positive uneradicated (patients without h. pylori eradicated or unsuccessful eradication) and h. pylori negative groups . For all patients, then, the healing rate of reflux esophagitis and remission of reflux symptoms were evaluated . Healed was considered if there was no esophageal mucosal break after treatment; unhealed indicated the presence of mucosal breaks . The healing rates among groups were comparedremission of reflux symptoms . The main reflux symptoms, including acid regurgitation, heartburn, and chest pain were scored before and after treatment . Healed was considered if there was no esophageal mucosal break after treatment; unhealed indicated the presence of mucosal breaks . The main reflux symptoms, including acid regurgitation, heartburn, and chest pain were scored before and after treatment . The test and t - test were used, respectively, for enumeration and measurement data . All statistical tests were two - sided, and a p <0.05 was considered statistically significant . Patients from digestive disease center of peking university third hospital, wu han union hospital, shanghai changhai hospital, and the first affiliated hospital, sun yat - sen university were enrolled between january 2007 and october 2010 . Included criteria were: almost 1870 years of age, typical reflux symptoms (heartburn, acid regurgitation and chest pain), and diagnosis of reflux esophagitis (los angeles [la] a - d degree). The la classification was used for the patients with reflux esophagitis, and those with reflux esophagitis la - a to la - d were all enrolled . Patients were excluded with: peptic ulcer, carcinoma of upper gastrointestinal tract, esophageal varices, zollinger ellison syndrome, esophageal stricture, barrett's esophagus, other severe gastrointestinal disease (for example ulcerative colitis), and accompanying severe diseases of other systems . Pregnant or lactating females, and patients who had undergone proton pump inhibitor (ppi) or bismuth therapy during the preceding 2 weeks were also excluded . Biopsy specimens for rapid urease test (rut) were obtained from biopsy forceps with a sterile needle and assessed immediately; the results were examined by an experienced observer blinded to the clinical details . The biopsy specimens for pathology were examined by an experienced pathologist after warthin starry (ws) staining . A sample was considered h. pylori positive with both rut and ws staining results positive . H. pylori negative samples showed negative results in both tests . When discordant results were obtained for rut and ws, the patients were excluded from the study . Biopsy specimens were examined by ws staining, and samples were considered h. pylori positive with positive ws staining . The severity of reflux symptoms was scored by the frequency and severity of the symptoms [table 1]. Scores of frequency and severity of reflux symptoms we divided a day into three periods: from breakfast to lunch, lunch to supper and supper to breakfast . If a reflux symptom appears in one, two or three periods, a score of one, two or three, was attributed, respectively . H. pylori positive patients were first randomly divided into eradicated and uneradicated groups by random envelope . In the eradication group, patients underwent h. pylori eradication for 10 days, then received esomeprazole 20 mg bid for the following 46 days . Two eradiation regimens were randomly selected in this study: the eac regimen consisted of esomeprazole 20 mg bid, amoxicillin 1.0 bid and clarimothin 0.5 bid; in the sequential regimen, esomeprazole 20 mg bid and amoxicillin 1.0 bid were administered for the first 5 days, while esomeprazole 20 mg bid, clarimothin 0.5 bid and tinidazole 0.5 bid were given for the following 5 days . In h. pylori positive uneradicated and h. pylori negative groups, afterward, all patients underwent gastroscopy again, and the ws staining test was repeated . Therefore, three groups were obtained: h. pylori positive eradicated, h. pylori positive uneradicated (patients without h. pylori eradicated or unsuccessful eradication) and h. pylori negative groups . Then, the healing rate of reflux esophagitis and remission of reflux symptoms were evaluated . Healed was considered if there was no esophageal mucosal break after treatment; unhealed indicated the presence of mucosal breaks . The healing rates among groups were comparedremission of reflux symptoms . The main reflux symptoms, including acid regurgitation, heartburn, and chest pain were scored before and after treatment . Healed was considered if there was no esophageal mucosal break after treatment; unhealed indicated the presence of mucosal breaks . The main reflux symptoms, including acid regurgitation, heartburn, and chest pain were scored before and after treatment . The test and t - test were used, respectively, for enumeration and measurement data . All statistical tests were two - sided, and a p <0.05 was considered statistically significant . There were 176 patients infected with h. pylori and 180 patients without h. pylori . For h. pylori positive patients, ninety - two patients showed successful eradication, while the remaining 30 still had the bacteria, indicating an eradication rate of 75.4% . Therefore, there were 92 and 84 patients in the h. pylori positive eradicated and h. pylori positive uneradicated groups, respectively . No significant difference in age, gender, height, and weight was obtained among groups [table 2]. General status of patients in different groups before treatment h. pylori: helicobacter pylori; sd: standard deviation . Before treatment, reflux symptom scores in the h. pylori positive eradicated and h. pylori positive uneradicated groups were 8.04 and 7.13, respectively (p = 0.062). There was no statistically significant difference in the la degree between the two groups (p = 0.307) [table 3]. Esophagitis grade and symptom scores in the h. pylori positive groups before treatment h. pylori: helicobacter pylori; sd: standard deviation . After treatment, reflux symptom scores of 0.22 and 0.14 were obtained for the h. pylori positive eradicated and h. pylori positive uneradicated groups, respectively (p = 0.588). The healing rate of reflux esophagitis were 80.4% and 79.8%, respectively, in the h. pylori positive eradicated and h. pylori positive uneradicated groups (p = 0.911) [table 4]. Healing rate and symptoms scores in the h. pylori positive groups after treatment h. pylori: helicobacter pylori; sd: standard deviation . Before treatment, reflux symptom scores in the h. pylori positive uneradicated and h. pylori negative groups were 7.13 and 7.18, respectively (p = 0.910), and no statistically significant difference in la degree between the two groups was obtained (p = 0.848) [table 5]. Esophagitis grade and symptom scores in the h. pylori positive uneradicated and h. pylori negative groups before treatment h. pylori: helicobacter pylori; sd: standard deviation . After treatment, reflux symptom scores in the h. pylori positive uneradicated and h. pylori negative groups were 0.14 and 0.21, respectively (p = 0.546). The healing rates of reflux esophagitis were 79.8% and 82.2%, respectively, in h. pylori positive uneradicated and h. pylori negative groups, respectively (p = 0.632) [table 6]. Healing rate and symptom scores in h. pylori positive uneradicated and h. pylori negative groups after treatment h. pylori: helicobacter pylori; sd: standard deviation . There were 176 patients infected with h. pylori and 180 patients without h. pylori . For h. pylori positive patients, ninety - two patients showed successful eradication, while the remaining 30 still had the bacteria, indicating an eradication rate of 75.4% . Therefore, there were 92 and 84 patients in the h. pylori positive eradicated and h. pylori positive uneradicated groups, respectively . No significant difference in age, gender, height, and weight was obtained among groups [table 2]. General status of patients in different groups before treatment h. pylori: helicobacter pylori; sd: standard deviation . Before treatment, reflux symptom scores in the h. pylori positive eradicated and h. pylori positive uneradicated groups were 8.04 and 7.13, respectively (p = 0.062). There was no statistically significant difference in the la degree between the two groups (p = 0.307) [table 3]. Esophagitis grade and symptom scores in the h. pylori positive groups before treatment h. pylori: helicobacter pylori; sd: standard deviation . After treatment, reflux symptom scores of 0.22 and 0.14 were obtained for the h. pylori positive eradicated and h. pylori positive uneradicated groups, respectively (p = 0.588). The healing rate of reflux esophagitis were 80.4% and 79.8%, respectively, in the h. pylori positive eradicated and h. pylori positive uneradicated groups (p = 0.911) [table 4]. Healing rate and symptoms scores in the h. pylori positive groups after treatment h. pylori: helicobacter pylori; sd: standard deviation . Before treatment, reflux symptom scores in the h. pylori positive uneradicated and h. pylori negative groups were 7.13 and 7.18, respectively (p = 0.910), and no statistically significant difference in la degree between the two groups was obtained (p = 0.848) [table 5]. Esophagitis grade and symptom scores in the h. pylori positive uneradicated and h. pylori negative groups before treatment h. pylori: helicobacter pylori; sd: standard deviation . After treatment, reflux symptom scores in the h. pylori positive uneradicated and h. pylori negative groups were 0.14 and 0.21, respectively (p = 0.546). The healing rates of reflux esophagitis were 79.8% and 82.2%, respectively, in h. pylori positive uneradicated and h. pylori negative groups, respectively (p = 0.632) [table 6]. Healing rate and symptom scores in h. pylori positive uneradicated and h. pylori negative groups after treatment h. pylori: helicobacter pylori; sd: standard deviation . H. pylori infection leads to chronic active gastritis with the potential for the subsequent development of peptic ulcer, malt lymphoma, and distal gastric cancer in some patients . Gerd is a common and costly disease in the community, with a negative impact on patients quality of life . The major mechanisms associated with gerd include decreased pressure and increased frequency of spontaneous relaxation of the lower esophageal sphincter, with normal or increased secretion of gastric acid . The relationship between h. pylori infection and gerd remains unclear, and the treatment of h. pylori in patients with gerd is highly controversial . The prevalence of h. pylori infection has been steadily decreasing while gerd incidence is increasing in developed countries, suggesting a possible protective role of h. pylori infection in the development of gerd . Other evidences include the following: the infection rate of h. pylori in gerd patients is much lower than in the general population; h. pylori is associated with the severity of gerd; the infection rate of h. pylori in severe gerd patients is much lower than those with mild gerd . Indeed, the prevalence of h. pylori was shown to be similar between gerd and other patients, and h. pylori was not related to the severity of gerd . Recently, japanese studies suggested that after h. pylori eradication, gerd symptoms are relieved, and life quality improved . The management of helicobacter pylori infection the maastricht iv / florence consensus report declares that h. pylori status has no effect on symptom severity, symptom recurrence and treatment efficacy in gerd . In the chinese guideline for h. pylori treatment, it was indicated that gerd incidence may be increased in some eastern countries after h. pylori eradication . Therefore, the question remains as whether for gerd patients with h. pylori infection, h. pylori eradication is necessary or useful . For the treatment of gerd, an agreement has been reached that ppi is the most important medicine . In the definition of montreal however, in case of h. pylori infection combined with gastroesophageal reflux, h. pylori eradication therapy is needed, which is confusing to the clinicians; this problem needs to be clarified as soon as possible . For most patients, but for different infection sites and types of gastritis, acid secretion may be decreased or increased after h. pylori infection . Based on this theory, some scientists have suggested that if acid secretion is increased by h. pylori infection, then the bacteria should be eradicated . On the other hand, so before h. pylori eradication, its effect on acid secretion should be first evaluated . But it is difficult in clinical practice for doctors . In a study by calleja et al . After 4 weeks treatment, the healing rate of reflux esophagitis was 86.6% in h. pylori positive patients . After 8 weeks treatment, the healing rates in positive and negative groups were 96.4% and 91.8%, respectively . The healing rates in the positive group were, therefore, higher than in the negative group, with significant differences . Also, the rate of gerd symptom relief was higher in h. pylori positive patients compared with negative patients . These findings suggested that based on the same treatment, a better outcome was obtained in h. pylori positive patients . In a prospective study by tefera et al . After 15 months of h. pylori eradication, there was no significant difference between acid secretion and gerd symptoms . In a study by de boer et al ., 1548 gerd patients underwent treatment with rabeprazole; there was no significant difference between h. pylori positive and negative patients in rates of gerd symptom relief . From the above studies, a large - scale study to compare the treatment outcome between h. pylori positive and negative comprehensively is needed . This was a multi - center, randomized, control, prospective study, with 356 patients enrolled . Based on the treatment of ppi, we explored the effect of h. pylori eradication on the treatment of reflux esophagitis . We analyzed the effect of h. pylori infection first, and patients in h. pylori negative and h. pylori positive control groups were compared . After ppi treatment, no significant differences in healing rates of reflux esophagitis and the severity of reflux symptoms were obtained, indicating that h. pylori infection has no significant effect on the treatment of reflux esophagitis . Patients with h. pylori infection were enrolled, and h. pylori eradicated and h. pylori positive uneradicated groups were compared . After the treatment with ppi, no significant difference was found in healing rates of reflux esophagitis and the severity of reflux symptoms . It was concluded that h. pylori eradication has no significant effect on the treatment of reflux esophagitis . Although we found that h. pylori eradication itself has no significant effect on erosive esophagitis treatment, the asia - pacific consensus on h. pylori infection mentioned that long - term use of ppi therapy causes increased risk of gastric atrophy, which is a precancerous lesion . For patients with gerd, ppi is used for a long time, therefore, h. pylori therapy is recommended . In this study, patients were followed - up for only 8 weeks, and we plan to continue follow - up for years in order to determine the changes by h. pylori eradication therapy; reflux esophagitis and relapse of reflux symptoms will be monitored, as well as gastric pathology.
Hyper immunoglobulin e syndrome (hies) is a rare primary immunodeficiency disorder characterized by elevated serum ige, dermatitis, and recurrent skin and lung infections . Three genetic etiologies of hyper ige have been identified: stat3, dock8, and tyk2 . Association with tyk2 deficiency has shown some reported cases of hyper ige with disseminated nontuberculous mycobacterial (ntm) infection . Microbial cultures of recurrent pulmonary infections show staphylococcus aureus, streptococcus pneumonia, and haemophilus influenzae more commonly while nontuberculous mycobacteria are secondary pathogens in pulmonary infections . Tuberculosis (tb) has been rarely reported in children with hies with sporadic report of miliary tb . A 15-year - old boy who had been previously diagnosed to have hies in view of recurrent skin pustules with dermatitis and elevated serum ige (ige = 3943 iu / ml [normal = 10180 iu / ml]) along with skin biopsy suggestive of lymphomatoid papulosis presented with recurrent abdominal pain and fever 3 months ago . Ultrasound (usg) of the abdomen showed multiple mesenteric lymphadenopathy, and mantoux test was positive (25 mm). He was started on four drugs for antituberculous therapy consisting of isoniazid (h), rifampicin (r), ethambutol (e), and pyrazinamide (z) for 2 months and then hr as continuation phase . However, the child stopped antituberculosis treatment (att) after taking it only for 3 months . He then again had pain in the abdomen and presented to us for further management . On examination, his weight was 24 kg, height was 134 cm, and he had pallor and papular dermatitis all over the skin . On systemic examination, he had mild tenderness in the periumbilical region . Investigations showed hemoglobin of 8.9 mg / dl, white blood cell count of 8100/cumm (58% polymorphs, 35% lymphocytes, 5% eosinophils, and 2% monocytes), erythrocyte sedimentation rate of 77 mm at the end of 1 h, and platelets of 238,000/cumm . Usg of the abdomen showed multiple mesenteric lymph nodes with largest being 1.6 cm 1.4 cm . His urine showed albuminuria with microscopic hematuria . A repeat serum ige was elevated (8856 he was started on four drugs for att (hrze), and urine was obtained for mycobacterium tuberculosis culture . Natural human immunity to mycobacteria group relies on the functional interleukin-12/23-interferon - gamma integrity of macrophages connecting to t - lymphocytes / natural killer cells . Patients with severe forms of primary immunodeficiency diseases have more profound immune defects involving this circuit, as seen in severe combined immunodeficiency, complete digeorge syndrome, x - linked hyper igm syndrome, cd40 deficiency, chronic granulomatous disease, and hies . A study shows that ntm infections are more common in hies patients with structural airway disease . In the absence of predisposing airway changes, ntm infections were not found in hies patients, suggesting that susceptibility to pulmonary ntm in hies may be more related to airway than immune dysfunction . An alternate explanation is the severity of immune dysfunction, through recurring infections, predisposes both to the severity of the structural lung disease and ntm disease . Our patient was diagnosed to have abdominal tb based on the presence of mesenteric nodes, a positive mantoux test, and previous response to anti - tb therapy . We do not have bacteriological confirmation of tb as the lymph node biopsy was not undertaken in our patient . Our patient did not have previous recurrent chest infections which could predispose to structural lung disease and thus he did not have pulmonary tb . The diagnosis of hies can be made based on a combination of clinical and laboratory findings for both types of hies . Clinically, patients usually present with recurrent skin and lung infections, in the form of abscesses, dermatitis, or pneumonia . Our patient had clinical features of skin involvement and elevated ige levels with recurrent skin infections suggestive of hies . We have not been able to do mutation analysis in our patient due to nonavailability of test . Antibiotic prophylaxis with trimethoprim - sulfamethoxazole is frequently used as prophylaxis against recurrent respiratory infections . Treatment of skin conditions such as eczema and skin infections is an important component of hies management . While there is no established guideline for the treatment of tb in primary immunodeficiencies, a case report of disseminated ntm in a patient with hies mentions that treatment with standard combination therapy for tb for a period of 12 months is indicated . Another case report of miliary tb in hies was successfully treated with first - line att drugs . The role of interferon - gamma, granulocyte - colony stimulating factor, or other immune modulators in hies is unproven . Bone marrow transplantation (bmt) is curative for autosomal - recessive hies with dock8 deficiency and it is recommended . Autosomal - dominant hies patients do well with intensive therapy and supportive care, and bmt is not recommended for those individuals.
A 30-year - old male patient received an above - elbow amputation about eight months prior to seeing us for an open fracture on the left radius and ulna due to trauma . He ended up transferring from one department to another department since his condition did not improve at all during treatment due to constant and severe pain after the amputation surgery . He kept complaining about cramping pain on his removed arm and electric - like pain occurring once every few minutes . He also said that he felt the entire shape of his removed arm, and it was medially rotated . Every day, he was prescribed gabapentine (2,400 mg), oxycodon (200 mg), and amitriptyline (25 mg), with other medications to control the pain . However, the degree of his pain relief was somewhat insignificant and the visual analog scale (vas) was 8 - 10 out of 10 . Other treatment methods, such as stellate ganglion block, thoracic sympathetic ganglion block, brachial plexus block, cervical transforaminal epidural block, and a subcutaneous infusion of ketamine, were also done . Lastly, spinal cord stimulation (scs) was done for the patient, but the treatment effect was very insignificant . Finally, we performed mirror therapy for the patient . He had to visit the hospital four times a week and went through a 15-minute treatment period . We had the patient feel the movement of his removed arm and hand just like his normal arm and hand moving through a mirror (fig . After a week passed, the patient said that he could feel his medially rotated arm was back to normal, and his vas level decreased to 7 out of 10 . One month later, he said that the previous cramping pain was almost gone and the phantom hand and arm returned to normal . At that time, his vas level was 5 out of 10 . After, three month from the initial therapy, he is doing a mirror therapy three to four times a week at home . However, the electric like pain remains and the vas usually is maintained at 4 out of 10 . He is under follow - up at our outpatient department with oxycodon decreased to 100 mg a day . No treatment for phantom limb pain has yet been clearly proven in terms of its effect . Drug therapy includes narcotic drugs, anti - epileptic medications, topical anesthesia, and analgesics . An infusion of ketamine, a n - methyl - d - aspartic acid receptor antagonist, was also introduced for phantom limb pain treatment . Meanwhile, non - drug therapy includes sympathetic ganglion block, transforaminal epidural block, peripheral nerve block, transcutaneous electrical nerve stimulation (tens), direct cortical or spinal cord stimulation, and mirror therapy, etc . Mirror therapy was unveiled by ramachandran and rogers - ramachandran in 1996 . Under this therapy, a patient is allowed to feel the imaginary movement of the removed body part behaving as normal body movement through a mirror . The mirror image of the normal body part helps reorganize and integrate the mismatch between proprioception and visual feedback of the removed body . Rizzolatti used a mirror neuron to explain the fundamentals of a mirror therapy . At first, a mirror neuron was found in the monkey premotor cortex, and later, rossi discovered that humans also have similar mirror neurons systems . A mirror neuron fires both when a person acts and when a person observes the same action performed by another . Then, the neuron mirrors the behavior of the other, as though the observer were itself acting . A mirror neuron provides observers with internally recognized experiences, making them understand other's behaviors, intentions, and emotional status . Therefore, while mimicking the behavior of the other, observers can experience not only the sensation, but also the similar emotion of the other . In this sense, a patient with phantom limb pain can feel the same sense or emotion of his / her normal body part by observing the mirror image . By doing so, it is expected to decrease pain by resolving conflict between motor intention, proprioception and visual system . A person without phantom limb pain and no amputations cannot feel these sensory experiences since the signs from a non - mirror neuron block the mirror neuron, while a patient with an amputation does not have this non - mirror neuron system operating . The visual observation can help feeling empathy, which explains how the mirror therapy works for a patient . It is reported that the therapy is more effective on deep somatic pain (e.g., pressure sense and proprioceptive pain) than on superficial pain (e.g., warmth sense and nociceptive pain). This is because deep tissues are responsible for integrating sensorimotor nerves as well as creating movements compared to superficial tissues . Recently, mirror therapy has used for not only patients with phantom limb pain, but also for patients with complex regional pain syndrome and strokes . Many studies indicate that mirror therapy is only effective for upper limb treatment, but it has potential as alternative treatment for pain that is difficult to control . In this study, mirror therapy resulted in dramatic pain relief for a patient with chronic phantom limb pain when other treatments such as medications, physical therapies, nerve blocks, nerve transformations did not work . Mirror therapy is expected to be widely used for the treatment of phantom limb pain since it is easy to use at both home and in outpatient departments.
Biopsies from the ampulla of vater are usually essential to diagnose premalignant and malignant lesions in asymptomatic or symptomatic patients . Acute pancreatitis is an extremely unusual complication following non - thermal biopsy of the ampulla without attempted cannulation . We present this case because of its rarity and the accompanying intense clinical manifestations without further severe consequences . A 51-year - old man underwent two gastroscopies over the prior 2 years with the indication of intermittent dyspepsia . On the last endoscopy, the ampulla of vater was described as bulging; biopsies were performed and histological examinations revealed mild chronic, nonspecific, inflammatory mucosal lesions of the ampulla and duodenum, with regional architectural disturbances, epithelial hyperplasia, accompanying mild nuclear stratification, and atypia, not fulfilling the diagnostic criteria for epithelial dysplasia . The patient was referred to our clinic with persistent, intermittent epigastric discomfort and pain, and we therefore scheduled a repeat examination in order to exclude an ampullary tumor . The extensive work - up after this episode did not reveal any aggravating factors for the embolism . In view of the results of the endoscopy, we advised the patient to discontinue acenocoumarol and changed to low molecular weight heparin (enoxaparin 40 mg / day). The patient was heterozygous for sickle cell disease (hemoglobin electrophoresis: hbs, 29.5%; hbf, 0.6%; hba2, 3.6%). A side - viewing scope (olympus tjf-145 video duodenoscope; olympus, tokyo, japan) was used with the patient under conscious sedation (midazolam 2 mg, propofol 60 mg). Eight biopsies were obtained from the area around the ampulla with a reusable oval - cup forceps (olympus fb-26n-1; olympus). No excessive bleeding one hour after the meal, the patient experienced an acute, severe (eight out of 10), persistent abdominal pain with recurrent, unrelieved vomiting . He contacted his family doctor but refused hospital admission and was monitored on an outpatient basis . On the second day, the patient reported less vomiting, but the pain was still intense and he additionally became febrile to 38.5c . He was advised to stop solid food intake and only take regular oral hydration . On day 3, the symptoms improved with no vomiting, milder abdominal pain, and a temperature less than 37.8c . Serum and urine amylase levels were 845 u / l (normal <100) and 8,500 u / l (normal <460), respectively . Kidney and liver tests showed normal results except for a slight elevation of the alanine aminotransferase level . Blood triglyceride level was 110 mg / dl (normal <150) and the calcium level was 9.2 mg / dl (normal 8.5 to 10.2). A second abdominal ultrasound was unremarkable, while contrast - enhanced abdominal computed tomography (ct) revealed mild enlargement of the pancreatic head and stranding of the surrounding retroperitoneal fat (fig . Eight tissue samples, with an approximate diameter of 1 to 3 mm each, were analyzed . Histological examinations revealed mild inflammatory mucosal lesions of the ampulla and duodenum, with regional gastric metaplasia, regional epithelial hyperplasia, and mild nuclear stratification, which did not meet the diagnostic criteria for epithelial dysplasia . The majority of periampullary neoplasms are malignant, whereas benign neoplasms account for less than 10% of the cases . Among benign lesions, adenomas are the most common, with a potential for malignant transformation . Although biopsies are essential in the diagnosis and follow - up of these lesions, the histological distinction between normal and pathological features of the ampullary mucosa is often difficult to make on endoscopically obtained samples . Acute pancreatitis is a well - recognized complication of endoscopic retrograde cholangiopancreatography, but is extremely rare after biopsy of the ampulla of vater without a previous attempt at cannulation . However, an asymptomatic increase in amylase concentration has been reported in up to 30% of cases . In the case we present, the diagnosis of acute pancreatitis was based on symptomatology in combination with the increase in serum and urine amylase levels, as well as the ct findings . To our knowledge, there are only three reported cases associating acute pancreatitis with endoscopic biopsies from the ampulla of vater . The first case was reported in a patient with gardner syndrome, who developed acute progressive periumbilical pain radiating laterally to the back 4 hours after endoscopy, with temperature increasing up to 39.2oc on the following day, high serum amylase levels, and a ct scan that showed pancreatic enlargement and diffuse stranding of the peripancreatic fat . Another case of severe acute pancreatitis was attributed to endoscopic biopsies of the minor papilla in a patient with pancreas divisum and liver metastases due to colon cancer . The third was also a case of severe pancreatitis with formation of a large pancreatic walled - off necrosis that was managed with ultrasonography - guided drainage . In all the aforementioned cases, abdominal pain was the presenting symptom and occurred 4 hours after sampling of the papilla . The clinical profile and course of our patient was similar to the case with gardner syndrome, with intense symptoms but still not a severe form of pancreatitis . Our patient was indeed on heparin up to 24 hours before the procedure, which in combination with the biopsy process could have led to the development of a small hematoma . Patients with sickle cell trait, especially african - americans, are more prone to manifest deep vein thrombosis and pulmonary embolism, although this remains a matter of debate . Vaso - occlusive phenomena have been described in patients with sickle cell trait (splenic infarction or sequestration, hyphema, etc . ). The occurrence of a thrombotic event would have led to ischemic pancreatitis, which is also a very rare condition and is usually associated with a major circulatory event such as hemorrhagic shock . Mucosal edema due to biopsies is the most plausible mechanism for pancreatitis in our case . We tried to direct the biopsies to the area around the orifice, but due to edema and bleeding after taking the first samples, it is sometimes very difficult to ensure that one or more biopsies are not directed towards the orifice . This same mechanism is also proposed by ishida et al . And morales and hixson, who reported cases of acute pancreatitis following endoscopic biopsy of the ampulla . Ishida et al . Additionally mentioned that the rise in the pancreatic duct pressure must have been low and of short duration, as no dilation of the pancreatic duct was observed . Despite the clear chronological association between sampling of the ampulla and acute pancreatitis in our patient no use of nonsteroidal anti - inflammatory drugs, paracetamol, proton pump inhibitors, antibiotics, or other commonly used medications were reported . Propofol has rarely been associated with pancreatitis if large doses are given in patients with coexistent hypertriglyceridemia . When we reviewed both the patient s older and recent blood tests, no evidence of pathological values for lipids and/or calcium was found . To our knowledge, there is no association of flumazenil with pancreatitis, whereas midazolam - induced pancreatitis reports has been scarcely reported and is not well - documented . Finally, it is worth mentioning that our patient recalled having substantial epigastric discomfort that lasted for 24 hours after a previous upper endoscopy when biopsies from the ampulla had been taken . One can assume that mild forms of pancreatitis may occur more often than is actually reported in the literature . In conclusion, we present a case of acute pancreatitis following biopsy of the ampulla . The case is significant for certain characteristics that have not been described in similar cases . Our patient s symptomatology was severe and not in parallel with the trivial laboratory and radiological pathology, and most importantly, with the excellent outcome . The patient s medical history entails features of unknown importance for the pathogenesis of pancreatitis (sickle - cell trait, previous use of anticoagulants, and repeat sampling), but these features may have contributed to the advent of pancreatitis; therefore, this case may be worth reporting for future reference . Finally, although pancreatitis after sampling of the ampulla is a rare complication, endoscopists should take it into consideration before the procedure and inform patients accordingly.
Leptomeningeal dissemination of tumor cells is frequently observed in terminal stage of malignant brain tumors, regardless of the primary origin . Occasionally, benign brain tumors, such as low - grade gliomas, spread to the subarachnoid space14). Recently, report of leptomeningeal dissemination of low - grade glioma is increasing as reflecting the common use of diagnostic magnetic resonance imaging (mri)15). It is known that approximately 5% of low - grade gliomas present with leptomeningeal dissemination at the time of diagnosis, and 7 - 10% of low - grade gliomas present with leptomeningeal dissemination at the time of disease progression15). One study reported the frequency of leptomeningeal dissemination via histology: pilocytic astrocytoma 3%, low - grade fibrillary astrocytoma 2%, gangliolioma 4%, mixed glioma 5%10). The frequency categorized by primary tumor site was as follows: cerebellum and brainstem 3%, cerebral cortex 1%, diencephalic region 7%, and spinal cord 1%10). We report an uncommon case involving a low - grade brainstem glioma with spinal dissemination, but without local recurrence . The patient was a 16-year - old boy who presented with diplopia, decreased vision, and gait disturbance of 2 months duration . On the neurologic examination brain mri demonstrated about 2.02.4 cm pontine lesion with low - signal intensity on t1 weighted image (wi) and high - signal intensity on t2wi without gadolinium enhancement (fig . Mr spectroscopy showed a slightly increased choline / creatinine ratio (1.07) and lactate peak, suggestive of a tumoral component and considered to be a pontine glioma . A biopsy was performed via an intrafacial triangle approach using the safe entry zone of the brainstem11). The histologic features were conspicuous cytoplasmic processes, mild nuclear pleomorphism, modest hyperchromasia, and no mitotic activity (fig . The immunohistochemical staining for glial fibrillary acidic protein was positive and the labeling index of ki-67 was 5 - 7% . Radiation therapy (total dose, 5400 cgy) was administered for the pontine lesion . The symptoms, especially the gait disturbance, were slightly improved after treatment; however, he still had a facial palsy and a limitation of lateral gaze . On the follow - up seven months after diagnosis, the gait disturbance was worsened and new - onset lower back pain developed . The brain mri showed a new lesion at the left cerebellopontine angle without an interval change in the primary lesion compared to the former images (fig . 3b). A spinal mri demonstrated leptomeningeal dissemination of the entire spine, primarily in the lumbosacral area (fig . Radiation therapy was started in the spinal seeding area (total dose, 3750 cgy). Subsequently, chemotherapy with a carboplatin plus vincristine regimen was administered . However, his general condition worsened with drowsy mentality . A brain mri showed an enlarged lesion associated with hemorrhage at the left cerebellopontine angle (fig . Two month later the patient was in a stuporous state and brain ct had a hemorrhagic lesion involving the left cerebellopontine angle and pre - pontine space which had increased in size (fig . Low - grade gliomas of the brain are common primary brain tumors, accounting for approximately 15 - 35% in most reported studies and characterized by slow progression and a median survival between 5 and 10 years2,6). Despite the long - term survival dissemination of tumor cells is the usual course of disease in primary central nervous system neoplasms; indeed, all types of brain tumors could undergo dissemination . This characteristic can be observed at the time of presentation or at the time of disease progression, primarily in medulloblastomas, germ - cell tumors, and high - grade gliomas8). Most reports have described neuraxis dissemination concurrent with or following recurrence at the primary site8,10). Civitello et al.3) reported that leptomeningeal dissemination was present in 6 of 162 children (3.7%) with low - grade gliomas: at diagnosis of the original tumor in one patient and with or following local relapse in four patients . One patient showed the leptomeningeal dissemination as a relapse sign but there was no examination for local recurrence . Spinal spread of primary intracranial tumors is more common in malignant brain tumors and some autopsy series have reported that such spreading occurs in 20 - 36% of supratentorial tumors and up to 60% of infratentorial tumors15,17). The incidence of tumor dissemination at the time of autopsy is probably higher than the clinical incidence of symptomatic metastasis . In low - grade gliomas, however, according to some reported studies, it has been estimated that 5% of low - grade gliomas are disseminated at the time of diagnosis, and 7 - 10% are detected along with the progressive course of the disease15). The increased use of mri studies might in turn increase the detection of disseminated disease . The underlying pathogenesis is unclear and there is no histologic evidence by which to differentiate disseminated disease from localized disease; however, some risk factors have been reported, such as invasion of the ependyma and fragmentation of tumor in contact with the csf5). The proximity of the tumor to the ventricular system and intra - operative ventricular entry may increase the risk of csf dissemination9). Depending on the location, tumor arising from the diencephalon appears to predispose to leptomeningeal dissemination10). On the contrary, one study suggested that intra - operative ventricular entry, primary tumor proximal to the ventricular system, and surgical resection could not increase the risk of tumor dissemination4). Dissemination of tumor cells via the csf is probably the primary mechanism for metastases within the central nervous system . Spread via the ventricular csf might be the most likely mechanism of tumor dissemination in hypothalamic tumors . The floor of the fourth ventricle was exposed via dissection of the tela choroidea and inferior medullary velum along the natural avascular plane on the left side . Seven months later, disseminated disease was detected in the cerebral subarachnoid space at the cerebellopontine angle and spinal cord, but not in the ventricular system . This pattern suggests direct shedding of tumor from the lateral foramen of the fourth ventricle through the operative tract into the subarachnoid space and transport via the csf to distant sites within the neuraxis . Patients with low - grade astrocytoma with neuraxis dissemination at the time of diagnosis have been reported to be a median survival of 15 months8). The course of disease varies from rapid progression, despite chemotherapy and radiation therapy, to prolonged stabilization . Treatment with carboplatin, alone or combined with vincristine, has been reported to achieve durable disease stabilization in patients with primary or recurrent low - grade gliomas, even in young children who were asymptomatic with widespread neuraxis disease7,12,13). The overall prognosis for patients with leptomeningeal dissemination at the time of recurrence appears to be worse than the prognosis for patients with a localized recurrence, and far better than for patients with a malignant brain tumor with dissemination3,10). However, the patient described herein had progressive features, even after radiation therapy and subsequent chemotherapy with carboplatin plus vincristitine . We report an uncommon case involving a low - grade brainstem glioma with spinal dissemination, but without local recurrence, and a progressive course associated with hemorrhage.
An 18-year - old man was referred to our clinic for the evaluation of blurred vision involving the left eye that had manifested four days previous . He had no specific medical, ocular, or trauma history . The subject's best - corrected visual acuity was 1.0 in the right eye and 0.9 in the left eye . Vf (central 10 - 2 sita - standard strategy) was measured with a humphrey field analyzer ii (zeiss - humphrey, san leandro, ca, usa) under continuous monitoring . The oct demonstrated disruption in the photoreceptor inner and outer segment (is / os) junction and undulation of the rpe with backscattering (fig . 2c), but the oct findings did not reveal improvement compared to the initial findings (fig . The oct demonstrated a recovery of continuity in the photoreceptor is / os junction, as well as decreased rpe irregularity with minimal backscattering (fig . Arpe is an acute, transient, foveal disturbance of unknown cause and which affects young adults . Because of scarce case reports and an infrequent prevalence of the disease, the diagnosis can be difficult without suspicion . Fluorescein angiography (fa) is a critical test for the differential diagnosis of arpe . Unfortunately, we could not obtain fa data because t he patient had signs of an anaphylactic reaction, such as dizziness and difficulty breathing, immediately after the fluorescein injection . Therefore, we made a diagnosis based on the fundus findings in combination with the oct and other characteristics of the disease . White dot syndromes, especially multiple evanescent white dot syndromes (mewds), should be considered in the differential diagnosis of arpe . White dot syndromes are characterized by multifocal white lesions and are accompanied by mild vitritis . Mewds differs from arpe in that the lesions are located outside the macula in the posterior pole, and electroretinogram (erg) findings are abnormal . The main lesions responsible for mewds cause damage to the photoreceptor outer segment but not the rpe . In our case, there was a single white dot lesion that was limited to the juxtafoveal region; also, erg, which was performed on the first visit, showed normal findings . In acute posterior multifocal placoid pigment epitheliopathy (apmppe), multiple, large placoid lesions start in the posterior pole and extend to the post - equatorial fundus, often accompanied by prodromal flu - like symptoms . Our patient's lesions were discrete clusters of a few subtle, small grey spots that resolved without scarring within three months after onset . In contrast, the lesions in patients with apmppe are replaced by rpe changes upon resolution . The oct demonstrated rpe involvement, which was similar to the previously reported oct findings . Based on these findings, other diseases were ruled out and the patient was diagnosed with arpe . Hsu et al . First described the oct findings of patients with arpe using time domain oct (oct3; carl zeiss) as a hyper - reflectivity involving the outer nuclear layer, photoreceptor, and rpe . Hyper - reflectivity has not yet been evaluated with sd - oct . In our case, we found a definite disruption in the is / os junction, as well as an undulation of the rpe according to sd - oct . Because sd - oct can provide better image resolution than oct3, we believe that the disruption of the is / os junction with sd - oct presented as a hyper - reflectivity of the outer retina with oct3 . As reported by hsu et al ., the thickness of the hyper - reflectivity decreased with increased resolution, and we found a decreased length of the is / os disruption based on the serial observations of the oct findings . The rpe irregularity also decreased as the symptoms improved and as the threshold of the visual field increased . On the basis of these findings, the morphologic changes in inflammation related to arpe do not seem to be permanent.
Congenital maternal hyperplasia (cah) is a group of autosomal recessive disease in which dysfunction of one of the five cortisol coding genes occurs and causes enzymatic defects in cortisol synthesis cycle from cholesterol . The most common form of the disease is 21-hydroxilase deficiency, which accounts for 90 - 95% of the cah cases . In the 1950 s, it was found that there were a small group of patients that developed hypertension and responded to glucocorticoid . Clinical manifestations of 11- - hydroxilase deficiency include, hypertension (roughly occurs in 75% of patients and generally diagnosed during childhood) and other signs related to overproduction of mineralocorticoids such as hypokalemia or muscle weakness, salt loss, and virilization . Signs of androgen excessive secretion include early closure of the epiphysis (short stature). The hypothalamic - pituitary - gonadal axis might cause amenorrhea or spermatogenesis disorders or hirsutism and acne . Schmid type is an autosomal dominant skeletal dysplasia in which the commonest form is metaphyseal chondrodysplasia . The schmid metaphyseal chondrodysplasia is characterized by short stature, but bone maturation process is normal . However, bowed legs, coxa vara, and specific metaphyseal changes are seen on radiographs . In this article, we report a case of a 4-year - old boy with cah and schmid metaphyseal chondrodysplasia . Our literature survey confirmed that it is the first reported case of coexistence of these two rare diseases . A 4-year - old boy with increased amount of pubic hair was referred to the children s endocrinology clinic . He was the first child of a family with normal growth and development until one year after birth . His parents were not related and there was no positive familial history . An increase in pubic and axillary hair developed four months later, he was treated for cah with 5 mg hydrocortisone, three times a day . Another problem with this child was gait disorder and bowed knee (figure 1). There was a similar history with his cousin, considering that the cousin was affected by skeletal dysplasia . Bowed knee in the patient with congenital adrenal hyperplasia . On clinical examination, doc level was 543 (ng / ml) and acth, dhea, 17-ohd, testosterone and androstenedione levels were higher than the normal limits . Biochemistry tests such as bun, cr, na, p, k, and alkp were normal . Laboratory test results radiological examination (plain plantar view and knee radiography) estimated the bone age to be about 10 years and 3 months . Finally, the patient was treated for precocious puberty due to 11- - hydroxilase deficiency and schmid metaphyseal chondrodysplasia . After 5 months, testosterone level decreased to lower than 10 ng / dl and dhea reached 27 mcg / dl . Schmid type metaphyseal chondrodysplasia is an autosomal dominant disorder and can be caused by various mutations in the col10a1 gene . Its diagnosis is hard due to the rarity of the disease and its similarity to rickets, particularly vitamin d resistant type . However, rickets and schmid can be differentiated with normal bone density and irregular dense zone in schmid chondrodysplasia . In our case, schmid metaphyseal chondrodysplasia was considered by clinical manifestation and was confirmed with radiology and laboratory tests . Bowed knee was the dominant characteristic sign and bone related biochemical tests such as ca, p and alkp were normal . The most important point in schmid disease is that over - treating by vitamin d, which can lead to toxicity, must be avoided . In some cases, congenital adrenal hyperplasias are a group of metabolic disorder diseases that lead to enzymatic defects in the biosynthesis of cortisol from cholesterol . Clinical features of 11--hydroxylase deficiency in childhood might be premature pubarche and accelerated bone age, all of which occurred in our patient . The exact diagnosis of 11--hydroxylase deficiency can be performed by the high basal levels of deoxycorticosterone and or 11-deoxycortisol serums or tetra - hydrometabolites, which might be found during a 24-hour urine test . This disorder should be considered in patients with elevated serum levels of acth, about three times higher than the 95 percentile predicted for patient s age . Its prevalence is 1 in 100,000 population and can be presented during childhood or adolescence . Because of familial marriage in iran, cah incidence is high . According to a study by qaemi et al, rohani showed that precocious puberty could be a manifestation of non - classical form of cah and its early diagnosis and treatment is important . In some cases, . Long - term complications of adrenal hyperplasia are short stature, infertility, gender identity disorders, and death . Female neonates with ambiguous genitalia and male with precocious puberty are in the highest risk category to develop htn because of the high secretion of deoxycorticosterone (doc). In our patient, mild htn was present . Although cah can be associated with other genetic disorders, but this is the first report on the association between cah and schmid metaphyseal chondrodysplasia . 11--hydroxylase deficiency is a rare disorder and must be considered in patients with precocious puberty and secondly presented with hypertension.
Biotinidase (ec 3.5.1.12) is the enzyme responsible for cleaving biocytin and recycling biotin from dietary protein - bound sources, . Profound biotinidase deficiency (less than 10% of mean normal serum activity) (omim #253260) is an autosomal recessively inherited metabolic disorder . Untreated individuals with profound biotinidase deficiency usually exhibit neurological and cutaneous symptoms with metabolic acidosis and organic aciduria, . Symptoms of the disorder can be markedly improved or prevented with pharmacological doses of oral biotin . However, if treatment is delayed, once vision or hearing problems or developmental delays occur, they are usually irreversible . All states in the united states and many countries screen their newborns for the disorder . The gene encoding biotinidase (btd) has been isolated and characterized, and over 150 mutations causing biotinidase deficiency have been identified . We now report the first microdeletion of btd that involves three of the four exons of the gene . This deletion further exemplifies the importance of performing microarray analysis or other methodologies for a deletion of btd when the enzymatic activity indicates lower activity than can be attributed to the mutations identified by dna sequencing . Microarray analysis was performed by whole genome chromosome prenatal reveal snp microarray (integrated genetics, labcorp specialty testing group). Dna sequencing of the biotinidase (btd) gene was performed by pcr amplification using primers and conditions described previously . All exonic and intron - exon boundaries of the btd gene were sequenced by prevention genetics (marshfield, wi). A non - consanguineous couple had prenatal diagnosis by microarray analysis for advanced maternal age . The results of the microarray analysis revealed a 26 kb interstitial microdeletion of chromosome 3p25.1 p25.1 (arr {hg19} 3p25.1 (15,674,11915,700,291) 1 . This heterozygous deletion is involves a major portion of the btd gene, including exons 24 (fig . 1). Based on this information, it was important to determine if the baby had a mutation on the other allele causing biotinidase deficiency . To determine the likelihood that the baby had such a mutation, the parents had their serum biotinidase activities determined . The father's activity was 5.5 nmol / min / dl (range of normal activity is 5.7 to 8.7 nmol / min / dl) and the mother's activity was 2.9 nmol / min / dl . These results indicated that the father had normal activity and did not have a mutation of btd and the mother had activity in the heterozygous range . In fact, microarray analysis of the parents revealed that the mother did have the microdeletion . The fetal dna was sequenced and did not reveal any other mutations or variants . At birth, the infant did not have biotinidase deficiency on newborn screening; however, the infant did not have serum enzymatic testing to confirm that her biotinidase activity was in the heterozygous range . We have previously reported a child with a contiguous gene deletion that involved three genes, including the btd gene . The child reported here is heterozygous for a microdeletion that only involves the btd gene . This deletion involves three of the four exons of the btd gene and is predicted to result in complete loss of biotinidase activity . This was confirmed by finding activity in the heterozygous range in the mother who also has the microdeletion . The reference laboratory that performed the microarray analysis indicates that they report deletions as small as 50 kb and they may report susceptibility genes when they are associated with clinical presentations that have a clear phenotype . However, many commercial laboratories that perform microarray analyses do not report microdeletions or duplications of less than 200 to 400 kb, unless the alteration involves a gene known to cause a dominant pathogenic disorder . In the instance reported here, the deletion is only 26 kb and an alteration of the involved gene, btd, which is only pathogenic as an autosomal recessive disorder . Therefore, it is possible that some or most laboratories would have not reported this deletion, unless they were specifically performing testing for biotinidase deficiency . If, for example, an individual has biotinidase activity in the profoundly biotinidase deficient range and has a missense mutation on one allele and a deletion on the other, sequencing would have only identified the missense mutation . If this scenario occurs in an asymptomatic, profoundly enzyme deficient infant identified by newborn screening, it is imperative to reconcile the low enzymatic activity with finding only a single mutation . There are occasions when enzymatic activity is lower than expected from mutation analysis . In these cases, it is precisely for this reason we have recommended that confirmatory enzymatic activities be performed on the proband, parents and an unrelated control, . If there is confidence that the reduced enzymatic is not due to poor sample storage, then it is important to consider the possibility that a microdeletion is present on the second allele to explain the lower enzyme activity . It is important to consider microarray analysis for a possible deletion in children identified as having enzymatic activity on newborn screening consistent with profound biotinidase deficiency, but only are found to have a single mutation by btd sequencing . The possibility of a deletion involving part or all of the btd genes must be considered in those children having enzymatic deficiency that is inconsistent with the results of their mutation analysis.
The two membranes of the nuclear envelope, the inner nuclear membrane (inm) and outer nuclear membrane (onm), are separated by a uniform distance of 3050 nm . To facilitate the import and export of cargos, the inm and onm meet at junctions containing nuclear pore complexes (npcs). Furthermore, the onm is contiguous with the er . In addition to its role in containing the genome, the nuclear envelope has been implicated in cell signaling and regulation of many important cellular functions, such as localization of nuclear proteins, organization of heterochromatin, and dna repair . The role of nuclear envelope defects in a wide - ranging collection of human diseases is drawing increased attention to this complex cellular structure . In addition to npcs, connections between the inm and onm are also formed by linkers of nucleoskeleton and cytoskeleton (linc) complexes, consisting of sad1p, unc-84 (sun) proteins in the inm interacting with klarsicht, anc-1, and syne homology (kash) proteins (also known as nesprins) in the perinuclear space (pns). Sun and kash proteins interact via conserved domains at their c - termini in the pns . Sun proteins interact with lamins in the nucleoplasm and kash proteins recruit cytoskeletal components to the onm . In this way, linc complexes stabilize the nuclear envelope against cytoplasmic forces and facilitate nuclear positioning in a variety of cellular processes, including cell division, establishment of cellular polarity, fertilization, cellular migration, and differentiation . Furthermore, linc complexes have been hypothesized to maintain the even spacing between the 2 membranes of the nuclear envelope . In caenorhabditis elegans somatic cells, the sun protein unc-84 interacts with the kash protein unc-83, which recruits microtubule motors kinesin and dynein to facilitate nuclear migration . In the early embryo, hypodermal precursors line up in 2 rows across the dorsal midline . As cells intercalate and elongate to form one row of 16 cells, the nuclei migrate contra - laterally to the opposite side of the dorsal midline . The hypodermal cells eventually fuse, and in the resulting syncytia, hypodermal nuclei are anchored evenly throughout . In unc-84 or unc-83 mutant embryos, the elongation and intercalation of cells proceeds normally, but the nuclei fail to move from their initial positions . As the embryo continues to develop, mutant nuclei that fail to migrate are passively pushed toward the dorsal midline by underlying muscle cell migrations and can be observed by dic microscopy in the dorsal cord of the l1 larva . In the adult hypodermal syncytia, after cell fusion, in unc-84 or anc-1 mutants, the nuclei are unanchored and are free to drift throughout the syncytia, often clustering in groups . Most sun proteins are large, with more than half of the protein residing in the pns . The c - terminal 200 amino acids contain the conserved sun domain, which trimerizes into a cloverleaf structure with the n - terminal stalk formed by a right - handed trimer coiled - coil . While the structure of the linker domain between the transmembrane domain and the trimeric coil is unknown, it has been proposed that the trimeric coiled - coil continuously extends to the transmembrane domain, forming a rod approximately 45 nm long . Such an extended coil structure would be suitable to span the space between the onm and the inm and serve as a " molecular ruler " to regulate even spacing of the nuclear envelope (fig 1a). Interestingly, several divergent sun proteins, including human sun3 - 5, which are restricted to the testis, and c. elegans sun-1, which is restricted to the germ line, are predicted to have much smaller luminal domains than those of their somatic counterparts . The prediction is that tissues expressing these sun proteins would display narrower nuclear envelope spacing (fig . 1b), but nuclear envelope spacing in those tissues has not been directly examined . Sun proteins are depicted in the inner nuclear membrane (inm) with their nucleoplasmic domain in yellow and their conserved sun domain in red . Sun domains bind kash proteins (blue) in the outer nuclear membrane (onm). Npc are nuclear pore complexes where the inner and outer nuclear membranes are connected . (a) the linker domains of human sun1/2 and c. elegans unc-84, between the trans - membrane span and the sun domain, are predicted to form trimeric rods that span the 4050 nm distance between the inner and outer nuclear membranes . (b) shorter sun proteins (human sun35 and c. elegans sun-1) are predicted to have shorter luminal domains and, as a result, narrower nuclear envelope spaces . (c) in the absence of linc complexes, lack of connection of the cytoskeleton to the nucleoskeleton is expected to cause the onm to separate from the inm . Sun proteins are depicted in the inner nuclear membrane (inm) with their nucleoplasmic domain in yellow and their conserved sun domain in red . Sun domains bind kash proteins (blue) in the outer nuclear membrane (onm). (a) the linker domains of human sun1/2 and c. elegans unc-84, between the trans - membrane span and the sun domain, are predicted to form trimeric rods that span the 4050 nm distance between the inner and outer nuclear membranes . (b) shorter sun proteins (human sun35 and c. elegans sun-1) are predicted to have shorter luminal domains and, as a result, narrower nuclear envelope spaces . (c) in the absence of linc complexes, lack of connection of the cytoskeleton to the nucleoskeleton is expected to cause the onm to separate from the inm . The hypothesis that linc complexes may serve as molecular rulers for nuclear envelope spacing was primarily based on electron micrographs of hela cells either depleted for sun1 and sun2 or expressing a soluble dominant - negative sun domain fragment, showing large distortions of the pns and 100 nm separation of the onm away from the inm (fig . However, hela cells in culture are in a very different environment than most nuclei in vivo . Because they make extracellular contacts to a hard surface, stresses at the cell surface can cause long - range force propagation, extending to the nucleus and beyond . As a result, nuclei are usually more spherical and are presumably not subject to the same intracellular forces . Unc-84 is the only sun protein expressed in somatic tissue, yet null mutants are viable and fertile . In unc-84(null) tissues, both unc-83 and anc-1 fail to localize, which allowed us to examine nuclei completely lacking linc complexes . High pressure freezing and electron microscopy techniques are well established in c. elegans, which allowed us to expand our observations to a variety of tissues and developmental stages . The defects of the unc-84(null) mutant can be fully rescued by an unc-84 transgene, which allowed us to express truncated forms of unc-84 as the sole somatic sun protein in an otherwise null background and to examine their effects on nuclear migration . We previously used this technique to locate the transmembrane domain in unc-84, as well as multiple sorting motifs in the n - terminus required for inm localization . The absence of linc complexes caused significant deformations of the nuclear envelope in hela cell culture, but whether a similar phenotype would be observed in an animal lacking linc complexes remained an open question . The hypothesis that linc complexes serve as molecular rulers suggests that a sun - less animal would display gross nuclear envelope defects in most or all tissues throughout development . We therefore carried out a series of experiments to characterize the role of the sun protein unc-84 in the architecture of the nuclear envelope . We began by examining pre - morphogenesis embryos, near the stage where unc-84 and unc-83 function together to move nuclei contra - laterally across the dorsal midline of the developing hypodermis . In most wild type nuclei, the pns is consistently 3050 nm in width, in agreement with previously published results . Surprisingly, nuclei from the null mutant unc-84(n369) strain are not significantly different than the n2 wild type laboratory strain . The pns of most unc-84 mutant nuclei are evenly spaced throughout, and are not significantly wider than wild type . Similarly, in the first larval stage, most nuclei, including those of the pharynx and hypodermis, have normal nuclear envelope spacing in both wild type and unc-84(n369) animals (fig . Therefore, in contrast to hela cells, where an absence of linc complexes results in extreme separation of the onm away from the inm, in most c. elegans tissues, removal of linc complexes has little to no effect on nuclear envelope spacing . Cells in the interior of the animal likely have less rigid cell - cell contacts than tissue culture cells, resulting in less overall strain to the cytoskeleton . Figure 2.unc-84 is required for even nuclear envelope spacing only in body wall muscle cells . (a) hypodermal larval nuclei in the unc-84(n369) animal do not display blebbing of the nuclear envelope . (b - c) however, large distortions of the nuclear envelope were observed at the ends of body wall muscle nuclei (arrows in b) and occasionally, along the sides (arrow in c). (a) hypodermal larval nuclei in the unc-84(n369) animal do not display blebbing of the nuclear envelope . (b - c) however, large distortions of the nuclear envelope were observed at the ends of body wall muscle nuclei (arrows in b) and occasionally, along the sides (arrow in c). Larva, the best candidates for a cell type with increased intracellular tension resulting from cellular shape changes are striated body wall muscles . In contrast to most other tissues where nuclei are nearly spherical, muscle nuclei are oblong, with the long axis oriented parallel to the muscle fibers . The pns of muscle nuclei are an even 4050 nm width all around the nuclei, although in some nuclei, the pns narrowed along the long axis and/or widened at the ends . We then examined unc-84(n369) muscle nuclei to determine if linc complexes play a role in stabilizing the nuclear envelope space in regions where forces appear to be highest (fig . Indeed, unc-84 mutant muscle nuclei have pns widths of 100500 nm . As with wild type nuclei, the largest distortions are at the ends of nuclei, although on occasion, smaller blebs are observed on the long axes (arrow in fig . 2c). Each nucleus was observed over multiple serial sections, which allowed for observation of the change in shape of the blebs section by section . In the z - plane, the blebs start out small, grow larger near the centers of nuclei, and are imperceptible at the bottom surfaces of nuclei . Therefore, body wall muscle is the only c. elegans tissue of the wide variety of embryonic and larval tissues we observed that produces large nuclear envelope distortions similar to those observed in hela cells . The observation that most nuclei in the unc-84(n369) display normal nuclear envelope spacing does not in and of itself contradict the hypothesis that linc complexes set the dimensions of the pns . Because unc-84 is required to recruit unc-83 to the onm, unc-84(n369) nuclei do not have unc-83 at the onm and therefore lack a direct connection to motors that pull on the nuclear envelope in wild type nuclei . A direct test of this hypothesis required the introduction of an abnormally sized sun protein that retains the ability to recruit and interact with kash proteins . The hypothesis predicts that the pns should then change to accommodate this mutant sun protein in all tissues where it is expressed . The majority of the linker domain of unc-84 in the pns between the trans - membrane span and the conserved sun domain bears little sequence similarity to other sun proteins . Additionally, a hybrid protein with the c. elegans unc-84 nucleoplasmic domain fused to the human sun1 luminal domain, which contains 2 predicted coiled - coil domains, functioned normally in hyp7 nuclear migration, suggesting the linker domain is functionally conserved from worms to mammals . These two findings are consistent with a model where the linker domain of unc-84 assumes a helical structure . Also consistent with this model is our finding that deletion of 15 amino acids immediately preceding the sun domain of unc-84, which correspond to the 15 amino acids of human sun2 that form the coiled stalk of the trimer, completely disrupts recruitment of unc-83 . Intriguingly, deletion of approximately 300 amino acids of the unc-84 linker domain (residues 556861 between the transmembrane span and the conserved sun domain; fig . 3a) nonetheless produced a functional unc-84 protein that was able to recruit unc-83 to the onm and move nuclei . This unc-84 truncation therefore provided the perfect tool to directly test the hypothesis that sun proteins set nuclear envelope spacing . The nuclear envelope spacing in embryos expressing unc-84(556861) is not noticeably narrower than wild type . Nor is there significant narrowing of pns width in the muscle nuclei of larvae . Figure 3.two hypotheses for how unc-84(556861) interacts with kash proteins without narrowing the perinuclear space . (a) schematics of full - length unc-84 and the functional truncation mutant unc-84(556861). (b) linc complexes may be spaced far apart and localized pinching together of the 2 membranes may not be resolvable by tem . (c) the luminal domain of unc-84(556861) may be fully extended to accommodate the normal 4050 nm perinuclear space . Two hypotheses for how unc-84(556861) interacts with kash proteins without narrowing the perinuclear space . (a) schematics of full - length unc-84 and the functional truncation mutant unc-84(556861). The transmembrane domain (tm) is depicted in black . (b) linc complexes may be spaced far apart and localized pinching together of the 2 membranes may not be resolvable by tem . (c) the luminal domain of unc-84(556861) may be fully extended to accommodate the normal 4050 nm perinuclear space . Our results suggest that rather than setting nuclear envelope spacing, sun proteins have evolved to span the distance between the inm and onm . The question remains: if sun proteins do not determine nuclear envelope spacing, what does? Npcs certainly play a role, as the perinuclear space narrows around npcs in nuclei without linc complexes . However, our results, as well as sun protein knockdown in tissue culture, indicate that npcs are not sufficient to maintain 4050 nm spacing away from the pores . The answer may lie in the nuclear envelope's closest relative the er . The membranes of the nuclear envelope share many properties with the er and the shape of the nuclear envelope echoes the shape of er . During interphase, er membranes are mostly in sheets, and polyribosomes are thought to provide the force to keep them flat . Er sheets also have a characteristic spacing, although it is observed to be approximately twice as large as that of the perinuclear space in mammalian cells . The morphology of er sheets is maintained by the luminal spacer climp63; overexpression of climp63 results in an increase of er sheets when overexpressed . Intriguingly, and in agreement with our results, depletion of climp63 does not, in fact, result in blebbing of er membranes . Rather, the intermembrane distance in the er is reduced by half and more closely approximates the nuclear envelope distance, which is unaffected by climp63 levels . This suggests that climp63 functions to expand the width of er sheets to make them bigger than the default state found in the nuclear envelope . As the onm is contiguous with the er, polyribosomes could similarly be maintaining its flatness in the absence of linc complexes . Chromatin could provide a similar force on the nucleoplasmic side of the nuclear envelope . In most tissues, these forces may be stronger than forces applied on the nucleus by the rest of the cell, such that even in the absence of sun proteins, the nature of the membranes is to lay flat and evenly spaced . Therefore, the even spacing of the nuclear envelope in the absence of linc complexes may be similar to the default spacing for er sheets lacking climp63 . Alternatively, there may be other inner nuclear membrane proteins with large luminal domains that mediate perinuclear spacing . By contrast, body wall muscle cells, or cells adhered to a dish, experience more mechanical strain than most in vivo cells . These forces are presumably strong enough to overcome the forces working to keep the nuclear envelope flat in the absence of linc complexes . Another question is if the distance between the onm and inm is an inherent property of the membranes, how can unc-84(556861) mutant interact with kash proteins without narrowing the pns? Membranes could be pinched inward in areas too small to be resolved by em (fig . If only a few, sparse linc complexes are required to move a nucleus (fig . 3b), the pinching that results might not be resolvable under the sectioning and electron microscopy conditions we used . Alternatively, the remaining 6080 residues could be fully stretched out . While the full - length unc-84 protein may contain a trimeric helical rod that spans the distance between the onm and inm, the unc-84(556861) mutant might not assume the same conformation . It is possible that instead of a rod, each linker region is an unordered polypeptide chain, with a greater capacity to extend than the full - length version (fig . If each residue could fully extend to 3.8 in an open peptide backbone, the remaining residues in the linker domain could reach 2025 nm . Adding on an additional 1415 nm (5 each for the onm and inm and 45 for the sun domain), gives a total of 40 nm, similar to the distances observed in nuclei expressing unc-84(556861). The observation that the absence of linc complexes causes nuclear envelope architecture defects in cells under strain could have implications for understanding some human diseases . In c. elegans, nuclear envelope defects one such disease, emery - dreifuss muscular dystrophy (edmd), is associated with mutations in several nuclear envelope proteins, including nesprin-1 and -2 and sun1 . Edmd patients display early tightening of the elbows, achilles tendons, and neck, followed by progressive muscle wasting in the limbs and associated dilated cardiomyopathy . The mechanism by which these mutations cause disease in a tissue - specific manner with variations from patient to patient is poorly understood . We compared the swimming motility of unc-84(n369) larvae, near the same stage where nuclear envelope defects were observed by tem, to wild type control animals . Using both manual and computational scoring techniques the unc-84(n369) notably, the motility defects we observed cannot be attributed to the loss of ventral cord neurons that has been previously described, as the required nuclear migration event in precursor (p) cells occurs at a later stage in development . Thus, we have identified a novel locomotion defect in the unc-84(n369) mutant consistent with a muscle contraction defect . As the structure of the muscle fibers in the mutant animal were not distinctly different from wild type, the nuclear envelope architecture defects could contribute in some way to the locomotion defects in live animals . However, a neuronal defect in a different cell lineage could lead to the locomotion disorder . A potential candidate is the lumbar ganglion neuron pvq, which is mispositioned in 15% of unc-84(n369) animals . Nonetheless, our results open a new area for exploration in better understanding the relationship between muscle disease and nuclear envelope architecture . Our work has clarified the prevailing model for the role of sun proteins in nuclear envelope spacing . Sun proteins are necessary to maintain the even spacing between the inm and onm in cells under high mechanical strain, but are not needed in other cells, where the inherent characteristics of er - derived membranes are sufficient to keep them flat and evenly spaced . We also have also uncovered a potential functional consequence of nuclear envelope architecture defects in muscle tissue . Further experiments are needed to better understand the nature of this connection and the role it might play in human disease . Specifically, examination of nuclear envelope ultrastructure in tissues from mouse muscular dystrophy models, as well as patient samples, should be informative . We thank erin tapley (uc davis), kent mcdonald (uc berkeley), and benjamin cain (uc davis), our co - authors on the original manuscript, for their wonderful scientific collaborations . Cain is supported by american cancer society illinois division postdoctoral fellowship pf-13 - 094 - 01-cgc.
Older women with breast cancer have poorer one - year relative survival than younger women and are more likely to be diagnosed with advanced stage of disease . Women over the age of 73 are not routinely invited for screening on the english national health service (nhs) breast screening programme; most women with breast cancer of this age group therefore present symptomatically . We have developed a brief intervention to promote early symptomatic presentation of breast cancer in older women (the promoting early presentation (pep) intervention). It is a scripted one - to - one intervention, delivered to an older woman in a positive, collaborative, and motivational style by a health professional, providing the knowledge, motivation, confidence, and skills to present promptly on discovering a breast symptom . The pep intervention increased breast cancer awareness fourfold compared with usual care for up to two years in a randomised controlled trial, in which it was delivered by research health professionals [5, 6], and the effect was sustained after three years (report in preparation). The nhs breast screening programme currently invites women aged 5070 for two - view mammography every three years and a national randomised controlled trial of inviting women aged 4749 and 7173 is currently under way . The final invited mammogram provides an opportunity to promote early presentation to women at increasing risk of developing breast cancer, but no longer routinely invited for screening, at whatever age that may be . By promoting early symptomatic presentation, the pep intervention may reduce stage of breast cancer at diagnosis and is unlikely to lead to overdiagnosis: among women aged 70 and over, breast symptoms are very likely to be due to breast cancer . We aimed to examine whether we could train nhs rather than research staff to deliver the pep intervention, whether quality of delivery could be maintained, and whether the effect on breast cancer awareness shown in the randomised controlled trial could be replicated in routine clinical practice . We piloted the pep intervention, which takes about five minutes, delivered by nhs mammographers as an integral part of the final invited mammogram appointment in four breast screening services . During 2011, we offered training to deliver the pep intervention to all 63 mammographers (both radiographers and assistant practitioners working in four breast screening services (cambridge and huntingdon; warwickshire, solihull and coventry; maidstone; medway)). The facilitator - led training involved two half - day group sessions, two to four weeks apart, plus practice sessions with performance feedback in - between provided by coaching radiographers . The training team, including the facilitator and coaching radiographers, assessed competence to deliver the pep intervention during and at the end of training by completing a checklist of quality criteria (see the appendix) during observed interventions . The coaching radiographer calculated a quality score for each intervention for content out of 33 and for style out of 6 and converted these to percentages . Having identified strong and weak areas of quality of delivery, the coaching radiographer undertook a performance feedback session with the mammographer . Twelve of the criteria were considered the most important (nine for content and three for style (marked essential and desirable on the checklist (see the appendix))) and so performance feedback focused mainly on these . We measured mammographers' confidence to deliver key messages about early presentation before and immediately after training, using a self - complete questionnaire including seven questions answered on a scale of 110 . We calculated mean scores out of ten for each question before and after training . During the implementation period (between three and six months in each service between may 2011 and february 2012), women attending for final mammogram were allocated longer appointments and offered the pep intervention on arrival . The pep intervention was delivered as an integral part of the final invited mammogram in the x - ray room . We implemented a quality assurance programme to ensure consistently high - quality delivery of the pep intervention . This involved a coaching radiographer assessing, for each mammographer, an audiotaped intervention every two weeks, and a directly observed intervention every two months, using the checklist of quality criteria (see the appendix). The coaching radiographer assessed quality and used this as the basis for a fifteen - minute performance feedback session as described in the section on training to deliver the pep intervention . Evaluating the effect of the pep intervention on breast cancer awareness involved the four pilot services and two comparison services which did not offer the pep intervention (norwich and norfolk and gateshead breast screening services). Women were sent information about the evaluation with their final invited screening appointment letter three weeks before their appointment . Mammographers invited eligible women to take part when they attended, and if they consented, they were asked to complete a short questionnaire . This measured knowledge of breast cancer symptoms, knowledge that the risk of breast cancer increases with age and of lifetime risk of breast cancer, reported breast checking, confidence to detect a breast change, and barriers to seeing a doctor with a health problem . Women were also asked to provide ethnic group, whether they lived with a husband or partner and age of leaving full time education . Breast cancer awareness data collection in the pilot services took place over may 2011 to april 2012 and in the comparison services over march 2011 to january 2012 . We compared change in breast cancer awareness over one month in women receiving the pep intervention in the pilot services with that of women in the comparison services . We assigned each woman taking part in the evaluation an index of multiple deprivation score (imd) (2007) based on the area of residence used in the census 2001 (higher scores indicate more socioeconomic deprivation: the imd summarises income, employment, health and disability, education and skills, housing, service access, living environment, and experience of crime, based on a range of routine data sources, for a geographical area). We examined demographic differences between women who received the pep intervention and women in the comparison services and between women who responded at one month and women who responded at baseline only . Women were considered breast cancer aware if they knew that risk of breast cancer increased with age, recognised five or more nonlump symptoms of breast cancer, and reported checking their breasts at least once a month . We used repeated measures logistic regression models to examine change in breast cancer awareness from baseline to one month comparing women who received the pep intervention with women in the comparison services, including only those who provided data at both time points . We examined the effect on the odds ratios of controlling for demographic differences between the groups . We carried out semistructured interviews with mammographers several weeks after training to gain their impressions of training, coaching, performance feedback and delivery of the intervention, and how they felt it had contributed to their professional development . The project received ethics approval from the cambridgeshire 1 research ethics committee (10/h0304/90). Thirty two mammographers started and 27 completed the training programme (five did not complete it because of health problems and family commitments). At the end of training, all 27 were delivering the pep intervention to a satisfactory level (70% or more for content and 50% or more for style). Mean confidence scores for all seven questions increased over the training period (figure 1). Eight hundred and thirty women were offered the pep intervention (25% of women attending for their final invited mammogram at the four services) and 551 (66%) took it up . Nineteen mammographers ultimately delivered these interventions eight were not able to for a variety of personal and service reasons . Quality of delivery was well maintained for these 19 mammographers: based on fortnightly assessments, mean scores for content never fell below 80% and mean scores for style never fell below 70% . In the pilot services, 511 women were asked to participate in the evaluation of breast cancer awareness; 495 (97%) agreed to take part and completed a baseline questionnaire . Four hundred and fifty seven (92%) women also completed the one - month questionnaire . In one of the comparison services, 880 (64%) women attending for final invited mammogram agreed to take part and completed the baseline questionnaire; 789 (90%) women also completed the one - month questionnaire . In the other comparison service, only 82 (36%) women attending for their final invited mammogram agreed to take part and completed the baseline questionnaire . This is likely to have been because mammographers found it difficult to recruit women due to ongoing service developments, in particular the introduction of digital mammography . We did not include the women attending this service in the analysis because of the low response rate . Women who responded at both time points were more likely to be white and slightly less likely to live in socioeconomically deprived areas than women who responded at baseline only (white: 98% versus 94%, p <0.001; median imd 11.1 versus 12.0, p = 0.03). Women who received the pep intervention were slightly older, less likely to be living with a husband or partner, more likely to have left school after the age of 18, and less likely to be living in socioeconomically deprived areas than women in the comparison service . Table 2 shows change in breast cancer awareness and confidence to notice a breast change in the women who received the pep intervention and the women in the comparison service . Women who received the pep intervention had a much greater increase in breast cancer awareness than the comparison group . The increase was seen for all components of the score: women who received the pep intervention were more likely to recognise five or more nonlump symptoms of breast cancer, to know that a 70-year - old woman was most at risk of breast cancer compared to a 30- or 50-year - old woman or a woman of any age and more likely to report checking their breasts at least once a month than women in the comparison service after one month . Women who received the pep intervention were also more likely to know the lifetime risk of breast cancer and to report being fairly or very confident that they would notice a change in their breasts at one month compared with the comparison service . Adjusting for age, living with a husband or partner, age left full time education, and imd made little difference to the odds ratios . Barriers to symptomatic presentation were relatively rarely reported by the women (table 3). The most frequently reported issues making it difficult to see a doctor with a health problem were feeling that they were bothering their doctor, finding it difficult to make an appointment and worrying that the doctor is too busy to listen to them . The pep intervention had limited influence on barriers; the only statistically significant differences were very small: women who received the pep intervention were less likely to report that finding it difficult to make an appointment, that the doctor was too busy to listen to them, and that it was physically difficult to get to the surgery than in the comparison group . Adjusting for age, living with a husband or partner, age left full time education, and imd made little difference to the odds ratios . In interviews, mammographers were very positive about training, coaching, performance feedback, and delivery of the pep intervention . They saw the pep intervention as extending their role, enhancing their professional development, and they particularly valued the opportunity it gave them to interact with their clients . We successfully piloted implementation of the pep intervention in four breast screening services: we trained nhs mammographers who delivered the intervention confidently to a high standard, and who were positive about its effect on their professional development . The intervention increased breast cancer awareness at one month from 4% at baseline to 38% at one month . The effect of the intervention on breast cancer awareness in routine clinical practice is of a similar size as achieved by the pep intervention delivered within the randomised controlled trial, which increased breast cancer awareness at one month from 2% at baseline to 33% at one month . Our study shows that the pep intervention is as potent after one month when delivered in routine clinical practice by nhs staff as when delivered by research radiographers with very close quality control in a randomised controlled trial . We were surprised at this finding: interventions, whether pharmacological or complex, are often less potent in routine clinical practice than in randomised controlled trials [1012]. There are many possible reasons for this, including that in randomised controlled trials the delivery of the intervention being tested is strictly controlled, and the participants are self - selected and more motivated with better potential for a positive outcome . The success of the pep intervention in routine clinical practice is likely to be due to a high level of mammographer motivation engendered by the training and coaching, and attributes of the intervention itself, which gives the mammographers an opportunity to communicate positively with their clients . . This may be at least partly because barriers were rarely reported by the women . I feel i am bothering my doctor, reported by about 11% of women . This is less than has been found in other studies of barriers to symptomatic presentation asking a similar question, which have found that over 30% of british people reported that worry about wasting the doctor's time might put them off seeing a doctor with a symptom that might be serious [14, 15]. Our evaluation of the pilot implementation of the pep intervention was not as methodologically robust as the randomised controlled trial: in the pilot, women were not randomly allocated to receive the intervention or not, so those receiving the intervention may have differed in many ways from those who did not, in the comparison services . However, adjusting for known differences between the women in the intervention and comparison services made little difference to the findings . Moreover, the effect size was so large that variation in outcome between the intervention and comparison services is unlikely to be due simply differences between the populations involved . Implementation was not complete: the services did not manage to offer the pep intervention to all women attending for final mammogram . This was because not all mammographers were trained to deliver the intervention, and implementation was limited by the capacity of existing clinics and availability of temporary staff to backfill the trained mammographers' time . Were the intervention to be implemented more widely, it would be necessary to expand capacity of the services to incorporate an extra five minutes for every woman attending for their final invited mammogram (about 1 in 7 mammograms delivered). Uptake of the intervention among women was good, suggesting that the intervention was an acceptable part of the mammogram appointment . Whether increasing breast cancer awareness will reduce breast cancer mortality the evaluation is ongoing and will, in due course, report the effect on breast cancer awareness at one year, self - referral for screening, symptomatic breast clinic attendances, and breast cancer mortality . There is indirect evidence that breast cancer awareness influences mortality: women who delay presentation in breast cancer are more likely to have poor awareness of symptoms, and delay in diagnosis is related to worse survival in breast cancer . The uk has worse breast cancer survival than many countries with good access to high - quality health care . We estimate that 7,00012,000 women in england delay presentation for> 3 months each year [16, 18]. These women have 7% lower 5-year survival than those with shorter delays . This suggests that at least 500 women in england will die because of delayed presentation each year (assuming a 5-year breast cancer survival of 80% in women who delay <3 months and 73% in those who delay> 3 months). Delivered to all women attending for their final invited mammogram on the nhs breast screening programme, at whatever age that may be, the pep intervention could contribute to improving cancer survival in england so that it is nearer to that achieved in similar countries.
A cross - sectional study was performed in 85 patients who were seen in a usual rheumatology outpatient clinic setting in daegu, korea, between may 2012 and july 2012, and who received a diagnosis of as according to the modified new york criteria . The study was reviewed and approved by the ethical review board of the institute prior to its initiation, and written informed consent was received from every patient prior to enrollment . Patients were excluded from the study if they were younger than 18 years and had another rheumatic disease in addition to as . Each patient seen in the clinic, with any diagnosis, completed a korean version of mdhaq, which was translated and validated by lee et al . Each patient with as also completed a basdai and basfi . An asdas and basmi were recorded by a health professional, either a metrologist or rheumatologist . Rapid3 is scored on the mdhaq as the 0- to 30-point sum of three 0- to 10-point patient - reported measures: physical function, pain, and patient global estimate of status . Other measures were scored as described in the literature (but not included in this report). Basdai includes 6 measures: fatigue, back pain, pain and swelling of peripheral joints, pain of enthesis, degree of morning stiffness, and duration of morning stiffness . Basfi includes 10 queries concerning physical function, scored as integers from 1 (easy) to 10 (impossible) on a likert scale . Asdas - esr (erythrocyte sedimentation rate) or asdas - crp (c - reactive protein) are calculated according to a formula that includes patient global status, spinal pain, spinal stiffness, fatigue, physical function, spinal mobility, and acute - phase reactant (esr or crp). Basmi includes 6 measures: occiput wall distance, modified schober test, lateral spinal flexion, chest expansion, cervical rotation, and intermalleolar distance . Statistical analyses were performed using stata 12.1 for windows (college station, tx). The shapiro - wilk w test was used to identify normality of distribution in the study population . Wilcoxon rank - sum test and spearman correlations were computed for each individual measure and total scores in the composite indices, including deletion of pain scores from each index . Patients were classified for disease activity / severity according to basdai (4 active vs <4 inactive), asdas (1.3 active vs <1.3 inactive), and rapid3 categories that have been developed for ra: more than 12, high; 6.1 to 12, moderate; 3.1 to 6, low severity (the term severity is used, as self - report scores do not necessarily distinguish activity from damage); and 0 to 3, remission . It is noted that these categories were developed in comparison to the disease activity score in 28 joints (das28) for ra and have not been validated in as, although they may be applicable to other rheumatic diseases . Cross tabulations, tests, and scatter plots were used to analyze statistical significance of possible associations between rapid3, basdai, and asdas . A cross - sectional study was performed in 85 patients who were seen in a usual rheumatology outpatient clinic setting in daegu, korea, between may 2012 and july 2012, and who received a diagnosis of as according to the modified new york criteria . The study was reviewed and approved by the ethical review board of the institute prior to its initiation, and written informed consent was received from every patient prior to enrollment . Patients were excluded from the study if they were younger than 18 years and had another rheumatic disease in addition to as . Each patient seen in the clinic, with any diagnosis, completed a korean version of mdhaq, which was translated and validated by lee et al . Each patient with as also completed a basdai and basfi . An asdas and basmi were recorded by a health professional, either a metrologist or rheumatologist . Rapid3 is scored on the mdhaq as the 0- to 30-point sum of three 0- to 10-point patient - reported measures: physical function, pain, and patient global estimate of status . Other measures were scored as described in the literature (but not included in this report). Basdai includes 6 measures: fatigue, back pain, pain and swelling of peripheral joints, pain of enthesis, degree of morning stiffness, and duration of morning stiffness . Basfi includes 10 queries concerning physical function, scored as integers from 1 (easy) to 10 (impossible) on a likert scale . Asdas - esr (erythrocyte sedimentation rate) or asdas - crp (c - reactive protein) are calculated according to a formula that includes patient global status, spinal pain, spinal stiffness, fatigue, physical function, spinal mobility, and acute - phase reactant (esr or crp). Basmi includes 6 measures: occiput wall distance, modified schober test, lateral spinal flexion, chest expansion, cervical rotation, and intermalleolar distance . Statistical analyses were performed using stata 12.1 for windows (college station, tx). The shapiro - wilk w test was used to identify normality of distribution in the study population . Wilcoxon rank - sum test and spearman correlations were computed for each individual measure and total scores in the composite indices, including deletion of pain scores from each index . Patients were classified for disease activity / severity according to basdai (4 active vs <4 inactive), asdas (1.3 active vs <1.3 inactive), and rapid3 categories that have been developed for ra: more than 12, high; 6.1 to 12, moderate; 3.1 to 6, low severity (the term severity is used, as self - report scores do not necessarily distinguish activity from damage); and 0 to 3, remission . It is noted that these categories were developed in comparison to the disease activity score in 28 joints (das28) for ra and have not been validated in as, although they may be applicable to other rheumatic diseases . Cross tabulations, tests, and scatter plots were used to analyze statistical significance of possible associations between rapid3, basdai, and asdas . Median age was 38.7 years; disease duration, 7.0 years; body mass index, 23.8 kg / m; level of formal education, 14.0 years; and hla - b27 positivity, 94.1% (table 1). Demographic and clinical characteristics of study patients rapid3 scores were correlated significantly with basdai (= 0.82, p <0.001), basfi (= 0.66, p <0.001), and asdas (= 0.76, p <0.001) (table 2, fig . ). These correlations were in the same range as correlations of basdai with asdas - esr (= 0.81) and asdas - crp (= 0.77) (fig . ). Spearman correlations of rapid3 with as disease - specific composite and individual measures including basdai, asdas, and basfi correlation plots comparing scores for rapid3 versus (a) basdai, (b) asdas - esr, and (c) asdas - crp with rapid3, and (d) scores for basdai versus asdas - esr . Note high correlations of these measures, in the same range for rapid3 versus as indices as for basdai versus asdas . Correlations of rapid3 were significant with individual responses on the basdai or basfi for spinal pain (= 0.75, p <0.001), spinal stiffness (= 0.74, p <0.001), fatigue (= 0.68, p <0.001), and physical function (= 0.66, p <0.001) (table 1). Correlations of rapid3 with physical measures of spinal mobility, or with acute - phase reactants esr and crp, were not statistically significant (table 2). Overall, 20, 25, 23, and 37 patients had rapid 3 scores indicating high, moderate, low severity, and remission, respectively, according to categories developed initially for ra . Interrater agreement of rapid3 remission or not in remission with asdas inactive or active was significant (= 0.486, agreement = 77.8%, p <0.001), but low between rapid3 remission or not in remission and basdai inactive or active (= 0.160, agreement = 45.6%, p = 0.002). However, all 20 patients with basdai scores of 4 or greater, indicating active as, had rapid3 scores of greater than 12, indicating high severity (table 3). Among 33 patients with asdas of greater than 1.3, indicating active disease, 26 (79%) had high severity according to rapid3 (table 3). The basdai, asdas, basfi, and basmi are standard disease - specific indices to assess the status of patients with as . These indices may include measures from patient self - report, a health professional, and laboratory tests and are collected feasibly in clinical trials and other clinical research, as well as in routine care at a few sites with a special interest in as . However, most routine clinical rheumatology care settings do not collect an index to assess ra quantitatively; it appears unlikely that a site would collect a basdai in as patients when rapid3 is not collected in ra patients . It is difficult to collect several different patient questionnaires from patients with different diagnoses in busy clinical settings . Furthermore, collection of several domains of asdas core data set measures and calculation of complex formulas for asdas are not feasible in all individual patients seen in routine care, despite an excellent web site for calculation (http://www.asas-group.org/research/asdas_calculator/asdas.html). By contrast, it is rather simple for the clinic receptionist to distribute the same questionnaire to each patient upon registration at the clinic . This practice facilitates completion of the questionnaire by the patient in the waiting area and helps the patient prepare for the visit, so the information is available to both patient and rheumatologist at initiation of the encounter . Scoring of rapid3 on an mdhaq requires 5 seconds, and rapid3 is useful in all rheumatic diseases in which it has been studied, including ra, systemic lupus erythematosus, psoriatic arthritis, gout, vasculitis, osteoarthritis, and as . Rapid3 is feasible in assessing disease severity and improvement in routine clinical of ra and is similar to das28 and clinical disease activity index (cdai) to distinguish active from control treatments in ra trials of adalimumab, abatacept, and certolizumab . The limited feasibility of collecting basdai and asdas as 1 of several questionnaires in routine care usually leaves laboratory tests as the only quantitative data available for clinical decisions in the medical records of most patients with as . However, laboratory tests often are not available at the time of the encounter and often are normal in patients with active clinical disease . Thus, improvement (or worsening) in as patients is characterized only by narrative descriptions, rather than by quantitative clinical data . First, it is cross sectional; longitudinal studies extending a recently published small series including as over a short period would be desirable in clinical trials and usual care . Second, low s between rapid3 and basdai, although statistically significant, were not as strong as might be ideal, explained in part by the presence of joint damage and/or concomitant fibromyalgia in patients who do not have active disease on physical examination . This phenomenon has been documented to be a significant issue with das28 and cdai in ra, in which patients with scores indicating moderate or severe activity did not have intensification of therapy, explained in large part by joint damage and fibromyalgia . In our study, all 21 patients with basdai of 4 or greater, indicating active disease, had rapid3 of greater than 12, indicating high severity . Rapid3 is thus effective to provide the most important goal of recognizing patients with severe disease activity who may require intensification of treatment, particularly in the era of anti tumor necrosis factor agents, although some of these patients may not require aggressive anti - inflammatory therapy . A third possible limitation is that rapid3 was not correlated significantly with metrology scores or laboratory tests . However, low correlations of patient indices with laboratory tests and imaging data are seen in ra . For example, a cdai is as effective to document changes in status as a simplified disease activity index, which includes an esr or crp . Of course, as - specific spinal flexibility measures are informative in clinical trials and other research and valuable in development of improved treatments, but may not be required in usual care . A fourth possible limitation is that highly significant correlations do not necessarily imply identity of information contained in 2 measures, as a line indicating near identity may not go through the origin at zero, but be shifted upward or downward . A shift upward is seen for correlations of rapid3 (as well as basdai) with asdas scores . Furthermore, correlations of = 0.8 are at a level seen in test - retest studies of the same measure, as high as seen in clinical medicine . Similar high correlations have been observed in 3 other settings in the united states, turkey, and norway, indicating great similarity of the information provided by the 2 questionnaires . The authors do not advocate at all that rapid3 should replace basdai, basfi, asdas, or basmi . Furthermore, rapid3 (or any index) must be supplemented by a standard medical history, physical examination, laboratory tests, and/or any other clinical information regarded as important by the rheumatologist in formulating clinical decisions . Collection of rapid3 in no way precludes collection of basdai, asdas, or any other index . Nonetheless, rapid3 is feasible in usual care and appears preferable to the absence of any quantitative clinical measure at all, as is the case for most as patients seen by rheumatologists at this time . The mdhaq / rapid3 might be added to research protocols to provide information to facilitate quantitative clinical assessment in routine care of patients with as.
Osteochondral lesions involve the articular cartilage and underlying subchondral bone, and most often affect the knee and the talus (1). Osteochondral lesions of the femoral head are uncommon, and few studies have reported the imaging findings of such lesions . Since this condition occurs in relatively young patients, and osteochondral damage puts joints at high risk of subsequent degeneration (2), timely recognition of this disorder is important for proper treatment . Herein, we review previous literature related to osteochondral lesions of the femoral head and 796report a case in which lesion involving the bilateral femoral heads was observed . This lesion manifested as subchondral cysts according to the initial radiography images, and led to further evaluation by computed tomography (ct) arthrography and immediately obtained magnetic resonance imaging (mri) scan that revealed overlying articular cartilage defects . A 20-year - old female patient was referred to our hospital from a local hospital . Her chief complaint was pain in both hips along with severe right side pain of approximately 2 months duration . Notably, however, she had been a taekwondo athlete in high school for several years . On clinical examination, plain radiography of the hip showed well - defined subchondral radiolucent lesions with a subtle sclerotic rim in the superior portion of the right femoral head (fig . The acetabuli in both sides of the hip displayed mild dysplasia with lower limit of normal (center - edge angle, 24 - 25 degrees). No signs of degeneration, such as joint space narrowing or juxta - articular sclerosis, were observed, except for subtle osteophyte around the head - neck junction of the right femur . Deformations of the femoral head, such as flattening, or the presence of crescent signs were also absent . Based on imaging findings, a provisional diagnosis of avascular necrosis with subchondral cysts could not be excluded . To further investigate the lesion, 1b, c) revealed several subchondral cysts that were hypointense on the t1-weighted images (repetition time [tr]/echo time [te], 374/20) and hyperintense on the t2-weighted fat - suppressed images (tr / te, 1500/29). The overlying portion in this region revealed a focal articular cartilage defect measuring about 10 15 mm in size, consistent with an osteochondral lesion . No surrounding bone marrow edema was observed, and no cartilage damage was noted on the opposite site of the acetabulum . The scintigraphy scan with 99 m technecium methylene diphosphonate showed no increased activity in the femoral head . On arthroscopy, chondral damage was noted in the superior portion of the femoral head . Follow - up plain radiography of the hip performed about a year later revealed subchondral cysts in the superior portion of the left femoral head (fig . The patient reported no incidence of trauma in the meantime, other than mild hip discomfort . 1f) scans of the left hip also showed several subchondral cysts, measuring about 14 13 mm in size, with associated overlying articular cartilage loss . Retrograde inspection of previous hip mri scans performed at the local referral hospital 2 months before the first visit to our hospital revealed lesions of the left femoral head with abnormal signal intensity, including hypointensity on the t1-weighted images and hyperintensity on the t2-weighted images (fig . This led us to hypothesize that the subchondral cyst lesion had progressed from a pre - existing lesion already present one year before . The chondral defect was confirmed by arthroscopy and core decompression with a bone graft was performed, as had been done for the right side . Osteochondral lesions involve articular cartilage and the underlying subchondral bone, and mainly affect the knee and the talus (1). Osteochondral lesions of the femoral head are rare and the prevalence is unclear (3). Detailed reports of the imaging findings of such lesions are also quite limited . Therefore, osteochondral lesions of the femoral head can be easily overlooked, unless radiologists are suspicious of osteochondral lesions that develop in the femoral region . Furthermore, osteochondral lesions of the femoral head are known to cause joint degeneration (2). We diagnosed the osteochondral lesion by identifying the accompanying overlying articular cartilage defects by ct arthrography and mri, which were performed for further evaluation . We excluded a diagnosis of avascular necrosis due to the absence of a double line sign or a crescent sign of the femoral head on mri . Moreover, no signs indicative of primary osteoarthritis, such as joint space narrowing or juxta - articular sclerosis in both the acetabulum and femoral head, were observed . The causes of osteochondral lesions have not yet been elucidated, and are likely to be multifactorial (4). One of the well - known causes of the osteochondral lesion of the femoral head is legg - calv - perthes disease . In this secondary lesion, however, separated subchondral bone with articular cartilage from the joint surface is the main feature, which is different than our case . Other etiologies of osteochondral lesions in the hip include previous infection, genetics, ischemia or trauma (5, 6). Many studies have reported damages incurred by repetitive traumas (7, 8, 9). (10) reported normal plain radiographic finding but mri detection of a subchondral lesion in an athlete with continuous hip pain, and suggested that although an athlete may not recall a specific incidence of past trauma, shearing injury of the cartilage by subluxation during joint rotation or impact damage caused by forces exerted by jumping could have occurred . (7) demonstrated that osteochondritis dissecans of the elbow can result from articular cartilage damage inflicted by repeated trauma . Nakamura et al . (6) reported an osteochondral lesion of the femoral head believed to have originated from repeated impact and high axial loads in a fencer . Our patient was previously an athlete, and so could have developed the osteochondral lesions by repetitive trauma, despite the lack of recollection of any particular past trauma . (10) also reported abnormal bone marrow signals from an osteochondral lesion on the femoral head . This lesion exhibited hypointensity on the t1-weighted images and hyperintensity on the t2-weighted images, along with cartilage surface irregularities of the medial portion . In our case, although the cartilage surface irregularities were not immediately apparent from the previous mri scans taken at the local referral hospital, lesions displaying hypointensity on the t1-weighted images and hyperintensity on the t2-weighted images were present in the left femoral head . An articular cartilage defect lesion accompanied by subchondral cysts was found about a year later, similar to the lesion previously observed on the right femoral head . We concluded that an osteochondral lesion was already present at the time of the previous mri scan, and that this lesion progressed to the lesion with subchondral cysts . It may often be necessary to distinguish osteochondral lesions from avascular necrosis in differential diagnoses . The articular cartilage of the femoral head has been shown to be unaffected in the majority of cases of avascular necrosis, whereas this cartilage is often damaged in osteochondral lesions . Moreover, avascular necrosis of the femoral head manifests itself as a somewhat broader lesion, often accompanied by serpiginous, low signal lines or crescent signs (11). On the other hand, osteochondral lesions often reveal smaller - sized areas with wedge - shaped signal abnormalities, or a narrow inlet with deep nest - shaped lesions, as seen in this case (12, 13). Patient age, in particular being younger, is also helpful in distinguishing this condition from avascular necrosis (10). 10) are thought to be indicative of lesions in the acute or subacute stage . In the chronic stage, however, a progressed degenerative bony lesion in the form of subchondral cysts, as seen in our case, or perhaps even a sclerotic change exhibiting hypointensity on the t1- and t2-weighted images, may be observed (6). Although some authors have reported that high uptake on the scintigraphy represents inflammation or lesion healing, no increased uptake was observed in our case, probably because the lesions are in the chronic stage . In addition, young patients, especially those with a previous trauma or a history of being an athlete, are potential candidates for osteochondral lesions . Radiologists should look for bony lesions associated with articular cartilage defects in the femoral head, which could be indicative of an osteochondral lesion, similar to the lesions that are known to occur in the knee or the talus.
Viral respiratory infections are a leading cause of illness and hospitalization in infants and children worldwide . In 2009 in the united states, more than 300,000 children were hospitalized as a result of infectious respiratory illness . Children with chronic medical conditions have an increased risk of morbidity and mortality from these viral respiratory infections [27]. In 2001, a new virus of the paramyxoviridae family called human metapneumovirus (hmpv) was identified . The virus, a member of the same subfamily as respiratory syncytial virus (rsv), has been credited as the etiology of non - rsv bronchiolitis due to its overlapping seasonal distribution [911]. Children with hmpv typically present with signs and symptoms of upper and lower respiratory tract infection such as fever, cough, tachypnea, and wheezing . In children less than five years of age, rates of hospitalization are similar between hmpv, parainfluenza, and seasonal influenza . Nebulized respiratory medications, such as albuterol and racemic epinephrine, have historically been administered in children presenting with viral lower respiratory infection . Recent evidence has led the american academy of pediatrics to recommend against the routine use of inhaled beta - adrenergic agents, such as albuterol, in the treatment of bronchiolitis . A recent cochrane database systematic review concluded that neither oxygen saturation nor length of hospitalization was improved by use of bronchodilators in bronchiolitis . Another cochrane review concluded that repeated dosing of racemic epinephrine in hospitalized children with bronchiolitis is also not recommended . While there is an abundance of data regarding the frequency of use and efficacy of beta - adrenergic agents and racemic epinephrine in children with rsv this study employed the following objectives: (1) to describe the clinical characteristics of patients admitted to the hospital with hmpv infection, (2) to determine the frequency of albuterol use in this population, and (3) to compare clinical outcomes, such as hospital length of stay (los), intensive care unit (icu) los, supplemental oxygen use, and mechanical ventilation, between hmpv patients who did and did not receive albuterol . We additionally hypothesized that there would be no relationship between the administration of albuterol and hospital los . All patients requiring admission to our institution with community - acquired hmpv infection between january and december 2010 were identified . We defined infection as the detection of hmpv from a nasopharyngeal or endotracheal specimen by polymerase chain reaction (pcr) testing obtained within the first 72 hours of admission . It was the general practice of our institution during the period of study to obtain respiratory viral pcr testing in any patient admitted to the hospital with infectious respiratory symptoms . The microbiology laboratory at our institution uses a multiplex respiratory viral pcr: the qiagen symphony rgq platform for nucleic acid extraction and amplification and a luminex system and test reagents for the determination of assay results . Other viruses identified with this pcr include rsv, influenza a and influenza b, adenoviruses, parainfluenza viruses 13, and human rhino / enteroviruses . We defined viral coinfection as the detection of other viral agents by pcr testing on the same specimen that detected hmpv . Patients with chronic medical conditions associated with increased morbidity and mortality with viral respiratory illness were identified [24, 16]. These conditions included chronic pulmonary conditions, cyanotic heart disease, prematurity, immunocompromised state, neuromuscular disorders, and hemoglobinopathies . Patients with a prior history of wheezing but without a formal diagnosis of asthma were identified from the electronic medical record, including notation of albuterol in home medications and documentation of wheezing in the past medical history or in any other section of clinician charting from that concurrent admission . Review of clinical and administrative databases and the electronic medical record was conducted by a single reviewer to establish hospital los, icu los, use of inhaled respiratory medications (e.g., albuterol, levalbuterol, racemic epinephrine, and/or ipratropium), supplemental oxygen use, chief complaint, and need for mechanical ventilation . Use of inhaled medications was determined through review of the patient's medication administration record for the patient's entire stay . An albuterol trial was defined as receipt of at least one dose of nebulized albuterol during admission and prolonged albuterol was defined as continued receipt beyond 24 hours of admission . These were children documented in the medical chart to clinically improve after albuterol based on a charting provider's evaluation of the patient . These providers included physicians, nurse practitioners, and respiratory therapists, and the clinical improvement was charted in varying words, including improvement in wheezing, decreased respiratory rate, or decreased work of breathing following a treatment . Based on admitting diagnosis in the electronic medical record, patients were also categorized as admitted with a primary respiratory or nonrespiratory complaint . Supplemental oxygen use was defined as the need for supplemental oxygen not associated with inhaled respiratory treatments . We calculated that a sample size of 120 patients would be needed to detect a difference in hospital length of stay of one day between groups (albuterol versus no albuterol) to achieve 80% power employing previously published hospital los estimates . All calculations were performed using stata / ic 12.1 (stata corporation, college station, tx). The median age was 16 months (interquartile range (iqr) 845 months) and 82 patients were female (40%). One hundred eighty - eight patients (91%) had positive specimens obtained prior to, or on the day of, hospital admission . Eighty - three percent of patients presented during the months of december to april with a peak incidence during the month of march (61 patients, 29%). The most common diagnoses on admission were acute respiratory tract infection (70%), status asthmaticus or asthma exacerbation (14%), vaso - occlusive crisis (4%), and seizure (3%). The characteristics of patients hospitalized with community - acquired laboratory - confirmed hmpv infection are listed in table 1 . Of the 31% of patients with a documented previous history of asthma or wheezing, one hundred percent of patients admitted with an asthma - related diagnosis received albuterol . Of the 179 patients admitted with a nonasthma - related diagnosis, 114 patients (64%) received albuterol . Forty - one patients (20%) were admitted to an icu, either pediatric or cardiac icu . In patients admitted to an icu, the median icu los was four days (iqr 110 days). One hundred forty - two patients (69%) received a trial of albuterol treatment . One hundred two patients (49%) received multiple albuterol treatments with albuterol during the first 24 hours of hospitalization . Thirteen patients (6%) required mechanical ventilation and four patients (2%) did not survive to discharge . We compared patient characteristics and clinical factors between patients who received albuterol and those who did not receive albuterol (table 2). There was no difference in hospital los between patients who received albuterol and those who did not . Patients with a previous diagnosis of asthma and those admitted with an asthma - related diagnosis were more likely to receive albuterol (both p <0.001). Patients with a nonasthma chronic medical condition were no more likely to receive albuterol than asthmatics and previously healthy patients (p = 0.09). Upon exclusion of asthmatics, those patients with a nonasthma chronic medical condition were more likely to receive albuterol than previously healthy children (p <0.001). Patients who received albuterol were more likely to have received supplemental oxygen (p <0.001). Male patients were more likely to receive albuterol (p = 0.03) than females, although, in our cohort, male patients were more likely to have a history of asthma (p = 0.01) than females . There was no correlation between age, sex, or chronic medical conditions with length of stay . Patients requiring supplemental oxygen had a longer length of stay (median 4 days versus 2 days, p <0.001). Children with prior wheezing or asthma had a shorter los (p = 0.001, median 2 days versus 3 days). Fifty - five percent of children in this study received prolonged albuterol therapy with a median duration of albuterol administration of four days (iqr 35 days). Patients receiving prolonged albuterol beyond 24 hours had a longer median hospital los (3 days versus 2 days, p = 0.025). This difference ceased to be significant after adjusting for supplemental oxygen use and asthma or prior wheezing . However, among the 80% of patients that did not require icu admission, prolonged albuterol use was associated with longer hospital los even after adjusting for supplemental oxygen use and asthma or prior wheezing (p = 0.046). Prolonged albuterol use was associated with an additional 1.7 hospital days after adjusting for supplemental oxygen use and asthma or prior wheezing among the cohort not requiring icu admission . Ninety - eight patients (47%) received at least one other nebulized respiratory medication: ipratropium (72 patients), racemic epinephrine (27 patients), dornase alfa (2 patients), and dexamethasone (1 patient). Patients who received a secondary nebulized respiratory medication twenty - eight (20%) of the 142 patients who received albuterol were described as albuterol responsive . As described above, previously healthy children, who comprised 43% of our cohort, were less likely to receive any albuterol than those with asthma or other chronic medical conditions . Forty - five percent of previously healthy children received albuterol trial, with 21% receiving prolonged albuterol . Other viruses recovered included human rhino / enterovirus (28 patients), adenovirus (9 patients), rsv (2 patients), influenza a (2 patients), and parainfluenza 3 (2 patients). There was no association between viral coinfection and any patient characteristics or clinical outcomes, most notably receipt of albuterol or hospital length of stay . Conclusion #1: the frequency of albuterol trial in children hospitalized with hmpv is high (69%). The frequency of prolonged albuterol use is high in children with hmpv (55%). Albuterol was administered to children both with and without a documented past history of wheezing or asthma . Our study observed widespread, frequent, and early (often within the first 24 hours of hospitalization) initiation of albuterol therapy to children with hmpv respiratory infection . This finding presents the question as to the reason for widespread bronchodilator use in this patient population . This may be true improvement but may also be transient due to concurrent administration of oxygen, other medication adjuncts (antipyretics, steroids, and antibiotics), and/or humidification in the setting of mask administration . Similarly, in a study of bronchodilator effects on airway reactivity in ventilated children with rsv bronchiolitis, only short term benefits were observed . In those children, acute improvement in airway reactivity, as evidenced by functional residual capacity, was suggested after albuterol treatments, but a clear translation of this improvement to long term outcomes was not observed . In children with a history of chronic illness and/or wheezing, their histories often reveal home use of albuterol or prior presentations with wheezing and albuterol . This correlates with our observation that 94% of patients with a history of wheezing or asthma received albuterol . This population, however, does not fully account for the widespread use of albuterol that we observed . Only 31% of patients in the study had a documented wheezing history, while 69% of patients received albuterol . Conclusion #2: the receipt of any albuterol was not associated with length of stay . We identified no difference in hospital los between hmpv patients who received albuterol treatment and those who did not . This finding was independent of length of albuterol treatment . In analysis of this conclusion, it is useful to consider an illness such as asthma, in which albuterol is a mainstay of treatment . One study of critically ill asthmatic patients found an association between continuous albuterol and a shorter los as compared to intermittent albuterol administration . In fact, another study looked at the specific gene known to play a role in response to beta-2 agonists such as albuterol . This study found asthmatic patients homozygous for glycine at that amino acid to have shorter icu length of stay as compared to other genotypes . Both studies suggest a role for albuterol in improvement of clinical outcomes in asthma; however, our results would not support a similar expectation in hmpv bronchiolitis . Results of our study are congruent with evidence on which the most recent aap guidelines were based in order to conclude that the routine use of bronchodilators does not have a clear benefit in treatment of bronchiolitis . Our results additionally highlight this finding in bronchiolitis caused specifically by hmpv . With attention to los and oxygen requirement, our findings do not support the routine use of albuterol in every patient with hmpv . Conclusion #3: prolonged albuterol use, beyond the first 24 hours of hospitalization, is associated with longer hospital length of stay . As the medical record does not include standardized markers of illness severity, we are unable to definitively rule out illness severity as a confounding variable of this result . One might propose that chronic illness may parallel illness severity, with those children having a longer los or being more likely to receive continued albuterol . We did not find an association between chronic medical conditions and length of stay, and a history of wheezing was actually found to be associated with shorter hospitalizations . A similar study in children with rsv also demonstrated an increased length of stay and duration of oxygen therapy in association with regular albuterol use . As more is known about hmpv and as pcr results become more rapidly available, knowledge of the pathogen may become useful to clinicians in decisions regarding albuterol continuation beyond 24 hours . Limitations of this study are acknowledged as this is a retrospective study where motivations to treat were not consistently clear to the researchers . Extensive chart review was conducted in order to capture those patient characteristics; however, information in electronic records is not fully standardized, nor does it guarantee completeness . Additionally, this study did not determine severity of illness in patients aside from intensive care admission and any potentially complicating chronic illness . Though we found high frequency of albuterol use in this patient population, we do not have a comparison frequency of use in all patients with respiratory illness in our institution . A patient's response to albuterol would be the ideal outcome to identify albuterol as the contributing variable, but we are limited to length of stay and prolonged albuterol administration to describe our population clinically . There is likely a difference between children experiencing an asthma exacerbation versus bronchiolitis secondary to hmpv infection, both in clinical motivators for albuterol and in disease pathophysiology . Due to the limitations of the patient chart as well as sample size, we were unable to analyze these two groups separately in regard to albuterol receipt . There is limited information on the period of time that hmpv continues to be shed in the nasopharynx of children . Small studies and case reports suggest a period of viral shedding of one to two weeks following acute infection [22, 23]. This prolonged shedding potentially confounds a positive pcr test for hmpv, especially in setting of asymptomatic patients . Our study, however, is predominately symptomatic patients with respiratory symptoms so severe as to require hospital admission . In conclusion, the overwhelmingly high frequency of albuterol administration in patients with hmpv infection, despite a lack of recommendations to support its practice in similar lower respiratory illnesses, is a key finding in this study . We found no clear association between hospital los and albuterol use in our hmpv infected patient population . In the future, research involving patients identified to have hmpv infection will be vital and useful in providing a smaller, etiology - specific cohort of patients with clear recommendations for management.
Two investigators (h.z . And s.z .) Carried out the comprehensive literature searches independently using the electronic databases medline, embase, and china national knowledge infrastructure without date and language restrictions . We used any possible combinations of relevant keywords of ppar (e.g., ppar, pparg, ppargamma, pro12ala, and p12a) polymorphism and each term designating t2dm - dn (e.g., type 2 diabetes, nephropathy, albuminuria, proteinuria, and esrd). Reference lists from relevant meta - analyses, systematic reviews, and clinical guidelines were also examined . The last quest was updated on 5 october 2011 . When more than one study of the same population was included in several publications, studies included in our meta - analysis had to meet the following inclusion criteria: 1) prospective cohort or case - control studies, 2) studies investigating the association of pro12ala polymorphism with t2dm - dn as the outcome, and 3) the control group with subjects who had t2 dm but were free of diabetic kidney disease . Information was carefully extracted from all eligible publications independently by two of the authors (y.t . And h.h . ). Discrepancies were adjudicated by the third reviewer (h.c .) Until consensus was achieved on every item . The following data were considered: author name, year and country of the study, ethnicity, genotyping method, and numbers of genotyped cases and control subjects . A total of 255 t2d subjects were selected from phase 2 of the chennai urban rural epidemiology study . The 255 individuals without microalbuminuria or proteinuria were randomly selected from all self - reported diabetic subjects (n = 1,529). Mohan s diabetes specialities centre, a tertiary center for diabetes in chennai, india . In all subjects, albumin excretion rate, measured by immuneturbidmetric assay, was at least 300 g / mg in at least two out of three fasting urine collections over a period of 3 months . The 141 proteinuric patients with albumin - to - creatinine ratio 300 g / mg were defined as case subjects . The clinical and biochemical characteristics of the data from our study had been described previously (11). Restriction fragment length polymorphism, and 10% of them were subjected to direct sequencing in order to confirm the quality of genotyping . The sequences of primers to genotype the pro12ala polymorphism of pparg were 5-gccaattcaagcccagtc-3 and 5-gatatgtttgcagacagtgtatc - agtgaaggaatcgctttccg-3 (12). The effect measures of choice were odds radio (or) for dichotomous variables and standardized mean difference (smd) for continuous parameters with their corresponding 95% cis . The departure of frequencies from those expected under hardy - weinberg equilibrium was assessed by goodness - of - fit tests in control subjects . We also calculated the quantity i that represented the percentage of total variation across studies . As a guide, values of i <25% may be considered low, and values> 75% may be considered high (14). The fixed - effects model was used when there was no heterogeneity among the included studies; otherwise, the random - effects model was used . In the absence of heterogeneity, the two methods provide identical results, because the fixed effects model, using the mantel - haenszel method, assumes that studies are sampled from populations with the same effect size, whereas the random - effects model using the dersimonian and laird method assumes that studies are taken from populations with different effect sizes, calculating the study weights both from interstudy and between - study variances, and considering the extent of variation or heterogeneity . The begg and mazumdar adjusted rank correlation test (15) and egger linear regression test (16) were used to provide diagnosis of the potential publication bias . The begg adjusted rank correlation test, a direct statistical analog of the visual funnel graph, tests for publication bias by determining if there is a significant correlation between the effect estimates and their variances and carries out this test by first standardizing the effect estimates to stabilize the variances and second by performing an adjusted rank correlation test based on kendall . The egger test detects funnel plot asymmetry by determining whether the intercept deviates significantly from zero in a regression of the standardized effect estimates against their precision . Sensitivity analyses were also performed to assess the stability of the results, namely, a single study in the meta - analysis was deleted each time to manifest the influence of the individual dataset to the pooled or (17). All meta - analyses were conducted using review manage, version 5.1 (the cochrane collaboration, oxford, u.k . ), whereas publication bias and sensitive tests were conducted using stata software (version 11.0; stata, college station, tx). Two investigators (h.z . And s.z .) Carried out the comprehensive literature searches independently using the electronic databases medline, embase, and china national knowledge infrastructure without date and language restrictions . We used any possible combinations of relevant keywords of ppar (e.g., ppar, pparg, ppargamma, pro12ala, and p12a) polymorphism and each term designating t2dm - dn (e.g., type 2 diabetes, nephropathy, albuminuria, proteinuria, and esrd). Reference lists from relevant meta - analyses, systematic reviews, and clinical guidelines were also examined . The last quest was updated on 5 october 2011 . When more than one study of the same population was included in several publications, studies included in our meta - analysis had to meet the following inclusion criteria: 1) prospective cohort or case - control studies, 2) studies investigating the association of pro12ala polymorphism with t2dm - dn as the outcome, and 3) the control group with subjects who had t2 dm but were free of diabetic kidney disease . Information was carefully extracted from all eligible publications independently by two of the authors (y.t . And h.h . ). Discrepancies were adjudicated by the third reviewer (h.c .) Until consensus was achieved on every item . The following data were considered: author name, year and country of the study, ethnicity, genotyping method, and numbers of genotyped cases and control subjects . A total of 255 t2d subjects were selected from phase 2 of the chennai urban rural epidemiology study . The 255 individuals without microalbuminuria or proteinuria were randomly selected from all self - reported diabetic subjects (n = 1,529). Mohan s diabetes specialities centre, a tertiary center for diabetes in chennai, india . In all subjects, albumin excretion rate, measured by immuneturbidmetric assay, was at least 300 g / mg in at least two out of three fasting urine collections over a period of 3 months . The 141 proteinuric patients with albumin - to - creatinine ratio 300 g / mg were defined as case subjects . The clinical and biochemical characteristics of the data from our study had been described previously (11). Restriction fragment length polymorphism, and 10% of them were subjected to direct sequencing in order to confirm the quality of genotyping . The sequences of primers to genotype the pro12ala polymorphism of pparg were 5-gccaattcaagcccagtc-3 and 5-gatatgtttgcagacagtgtatc - agtgaaggaatcgctttccg-3 (12). The effect measures of choice were odds radio (or) for dichotomous variables and standardized mean difference (smd) for continuous parameters with their corresponding 95% cis . The departure of frequencies from those expected under hardy - weinberg equilibrium was assessed by goodness - of - fit tests in control subjects . We also calculated the quantity i that represented the percentage of total variation across studies . As a guide, values of i <25% may be considered low, and values> 75% may be considered high (14). The fixed - effects model was used when there was no heterogeneity among the included studies; otherwise, the random - effects model was used . In the absence of heterogeneity, the two methods provide identical results, because the fixed effects model, using the mantel - haenszel method, assumes that studies are sampled from populations with the same effect size, whereas the random - effects model using the dersimonian and laird method assumes that studies are taken from populations with different effect sizes, calculating the study weights both from interstudy and between - study variances, and considering the extent of variation or heterogeneity . The begg and mazumdar adjusted rank correlation test (15) and egger linear regression test (16) were used to provide diagnosis of the potential publication bias . The begg adjusted rank correlation test, a direct statistical analog of the visual funnel graph, tests for publication bias by determining if there is a significant correlation between the effect estimates and their variances and carries out this test by first standardizing the effect estimates to stabilize the variances and second by performing an adjusted rank correlation test based on kendall . The egger test detects funnel plot asymmetry by determining whether the intercept deviates significantly from zero in a regression of the standardized effect estimates against their precision . Sensitivity analyses were also performed to assess the stability of the results, namely, a single study in the meta - analysis was deleted each time to manifest the influence of the individual dataset to the pooled or (17). All meta - analyses were conducted using review manage, version 5.1 (the cochrane collaboration, oxford, u.k . ), whereas publication bias and sensitive tests were conducted using stata software (version 11.0; stata, college station, tx). Out of 486 potentially relevant articles retrieved from electronic databases and reference lists, 17 case - control studies met the inclusion criteria (610,1827). An additional one set of data from our study was included . A flow diagram of search and selection is shown in supplementary fig . Fourteen datasets had albuminuria evaluated, two esrd, and three unrestricted dn (supplementary table 3). A total of 3,361 t2md - dn and 5,825 t2md subjects without dn were included . Additionally, there were five (7,19,21,24,25) and four studies (7,19,21,27) available to identify the effect of pro12ala polymorphism on the levels of serum creatinine (scr) or albumin excretion rate (aer) in t2 dm patients, respectively . The eligible data sets were obtained from 11 english - language and 4 chinese - language articles . Of these data sets, nine focus on a caucasian (five for italian and four for others), and the other nine focus on an asian population (five for chinese and four for others). Given the low frequency of ala allele, even in some studies, ala homozygous individuals are absent, and only the dominant model was investigated, comparing ala carriers to pro / pro . We also assessed the deviation of hardy - weinberg equilibrium in control subjects, and the results demonstrated that all the genotype distributions in the control groups had high goodness - of - fit . The methods used for measuring scr or aer in both case and control arms were the same . The other detailed characteristics of included studies were summarized in supplementary table 1 . In the case of t2 dm subjects without complications, no significant impact of the pro12ala polymorphism on the risk of proteinuria was observed, as ors (95% ci) were 0.98 (0.581.67), 3.64 (0.3240.6), and 1.04 (0.621.75) for pro / ala versus pro / pro, ala / ala versus pro / pro, and ala carriers versus pro / pro, respectively . Overall, the pro12ala polymorphism was found to be significantly associated with decreased t2md - dn risk (or 0.76 [95% ci 0.610.93]) (fig . 1). Both the cochran q test and estimate of i revealed significant heterogeneity among the constituent studies (ph = 0.03; i = 42%). Stratified analyses of ala carrier versus pro / pro and nephropathy susceptibility of t2 dm patients forest plots of the meta - analysis for pro12ala polymorphism of ppar associated with nephropathy in t2 dm patients . To avoid the influence of heterogeneity among the included studies, subgroup analyses were distinctively carried out for each stage of dn and ethnic group . In the analysis stratified by stage of dn, the significantly decreased risk of albuminuria (e.g., microalbuminuria and macroalbuminuria) was observed (or 0.72 [95% ci 0.580.89] for albuminuria; 0.38 [0.250.57] for microalbuminuria; and 0.64 [0.490.82] for macroalbuminuria), but there was no statistically significant association between pro12ala polymorphism and esrd . Furthermore, in the analysis stratified by ethnicity, significant association was detected between the polymorphism and dn risk in caucasian (0.68 [0.510.90]) but not in asian populations (0.85 [0.621.17]). Significant heterogeneity was eliminated in most of the subgroup analyses but the asian groups (ph = 0.05; i = 48). The details are also listed in table 1 . To test the stability of the pooled results, one - way sensitivity analyses of the pooled ors and when omitting each dataset in the overall meta - analysis, the pooled ors were always persistent (fig . Begg and egger tests were used to provide diagnosis of the potential publication bias, and no evidence of publication bias was found in the overall analysis (pbegg = 0.150; pegger = 0.085) (table 1 and fig . 3). Evidence also suggested no publication bias in the subanalyses mentioned above, but one for the association between ppar pro12ala and albuminuria (pbegg = 0.063; pegger = 0.036). However, after getting rid of one dataset each by caramori (24), de cosmo (7), pollex (21), or li (19), the existing bias disappeared (pbegg = 0.100, pegger = 0.053; pbegg = 0.100, pegger = 0.060; pbegg = 0.100, pegger = 0.089; and pbegg = 0.100, pegger = 0.056, respectively), whereas the significant ors were persistent . The persistent result indicated that the emerging publication bias did not decrease the reliability of the result . One - way sensitivity analysis of the pooled ors and 95% ci for the overall analysis, omitting each dataset in the meta - analysis . The se is plotted against a logarithmic scale of the or [log(or)]. We observed that levels of scr were significantly associated with the pro12ala polymorphism of ppar in t2 dm subjects (supplementary fig . 2, bottom). Overall, the smd for scr was statistically significant (smd = 0.12; 95% ci 0.23 to 0.01; z = 2.13, pz = 0.03). There was no significant heterogeneity among the trials (ph = 0.52; i = 0%). The effects of the polymorphism on the level of aer were all significant in the four included trials (supplementary fig . The synthetic smd for aer was significant (smd = 3.04; 95% ci 4.90 to 1.18; z = 3.20, pz = 0.001). However, the heterogeneity among eligible studies (ph <0.001; i = 99%) was significant . It is noteworthy that a significant decline in aer was detected in the individuals with 12ala, whereas no evidence of publication bias was observed (supplementary fig . Out of 486 potentially relevant articles retrieved from electronic databases and reference lists, 17 case - control studies met the inclusion criteria (610,1827). An additional one set of data from our study was included . A flow diagram of search and selection is shown in supplementary fig . Fourteen datasets had albuminuria evaluated, two esrd, and three unrestricted dn (supplementary table 3). A total of 3,361 t2md - dn and 5,825 t2md subjects without dn were included . Additionally, there were five (7,19,21,24,25) and four studies (7,19,21,27) available to identify the effect of pro12ala polymorphism on the levels of serum creatinine (scr) or albumin excretion rate (aer) in t2 dm patients, respectively . The eligible data sets were obtained from 11 english - language and 4 chinese - language articles . Of these data sets, nine focus on a caucasian (five for italian and four for others), and the other nine focus on an asian population (five for chinese and four for others). Given the low frequency of ala allele, even in some studies, ala homozygous individuals are absent, and only the dominant model was investigated, comparing ala carriers to pro / pro . We also assessed the deviation of hardy - weinberg equilibrium in control subjects, and the results demonstrated that all the genotype distributions in the control groups had high goodness - of - fit . The methods used for measuring scr or aer in both case and control arms were the same . In the case of t2 dm subjects without complications, the mean duration of diabetes was 6.6 years . No significant impact of the pro12ala polymorphism on the risk of proteinuria was observed, as ors (95% ci) were 0.98 (0.581.67), 3.64 (0.3240.6), and 1.04 (0.621.75) for pro / ala versus pro / pro, ala / ala versus pro / pro, and ala carriers versus pro / pro, respectively . Overall, the pro12ala polymorphism was found to be significantly associated with decreased t2md - dn risk (or 0.76 [95% ci 0.610.93]) (fig . 1). Both the cochran q test and estimate of i revealed significant heterogeneity among the constituent studies (ph = 0.03; i = 42%). Stratified analyses of ala carrier versus pro / pro and nephropathy susceptibility of t2 dm patients forest plots of the meta - analysis for pro12ala polymorphism of ppar associated with nephropathy in t2 dm patients . To avoid the influence of heterogeneity among the included studies, subgroup analyses were distinctively carried out for each stage of dn and ethnic group . In the analysis stratified by stage of dn, the significantly decreased risk of albuminuria (e.g., microalbuminuria and macroalbuminuria) was observed (or 0.72 [95% ci 0.580.89] for albuminuria; 0.38 [0.250.57] for microalbuminuria; and 0.64 [0.490.82] for macroalbuminuria), but there was no statistically significant association between pro12ala polymorphism and esrd . Furthermore, in the analysis stratified by ethnicity, significant association was detected between the polymorphism and dn risk in caucasian (0.68 [0.510.90]) but not in asian populations (0.85 [0.621.17]). Significant heterogeneity was eliminated in most of the subgroup analyses but the asian groups (ph = 0.05; i = 48). The details are also listed in table 1 . To test the stability of the pooled results, one - way sensitivity analyses of the pooled ors and when omitting each dataset in the overall meta - analysis, the pooled ors were always persistent (fig . 2). Begg and egger tests were used to provide diagnosis of the potential publication bias, and no evidence of publication bias was found in the overall analysis (pbegg = 0.150; pegger = 0.085) (table 1 and fig . 3). Evidence also suggested no publication bias in the subanalyses mentioned above, but one for the association between ppar pro12ala and albuminuria (pbegg = 0.063; pegger = 0.036). However, after getting rid of one dataset each by caramori (24), de cosmo (7), pollex (21), or li (19), the existing bias disappeared (pbegg = 0.100, pegger = 0.053; pbegg = 0.100, pegger = 0.060; pbegg = 0.100, pegger = 0.089; and pbegg = 0.100, pegger = 0.056, respectively), whereas the significant ors were persistent . The persistent result indicated that the emerging publication bias did not decrease the reliability of the result . One - way sensitivity analysis of the pooled ors and 95% ci for the overall analysis, omitting each dataset in the meta - analysis . The se is plotted against a logarithmic scale of the or [log(or)]. We observed that levels of scr were significantly associated with the pro12ala polymorphism of ppar in t2 dm subjects (supplementary fig . 2, bottom). Overall, the smd for scr was statistically significant (smd = 0.12; 95% ci 0.23 to 0.01; z = 2.13, pz = 0.03). There was no significant heterogeneity among the trials (ph = 0.52; i = 0%). The effects of the polymorphism on the level of aer were all significant in the four included trials (supplementary fig . The synthetic smd for aer was significant (smd = 3.04; 95% ci 4.90 to 1.18; z = 3.20, pz = 0.001). However, the heterogeneity among eligible studies (ph <0.001; i = 99%) was significant . It is noteworthy that a significant decline in aer was detected in the individuals with 12ala, whereas no evidence of publication bias was observed (supplementary fig . Overall, the pro12ala polymorphism was found to be significantly associated with decreased t2md - dn risk (or 0.76 [95% ci 0.610.93]) (fig . 1). Both the cochran q test and estimate of i revealed significant heterogeneity among the constituent studies (ph = 0.03; i = 42%). Stratified analyses of ala carrier versus pro / pro and nephropathy susceptibility of t2 dm patients forest plots of the meta - analysis for pro12ala polymorphism of ppar associated with nephropathy in t2 dm patients . To avoid the influence of heterogeneity among the included studies, subgroup analyses were distinctively carried out for each stage of dn and ethnic group . In the analysis stratified by stage of dn, the significantly decreased risk of albuminuria (e.g., microalbuminuria and macroalbuminuria) was observed (or 0.72 [95% ci 0.580.89] for albuminuria; 0.38 [0.250.57] for microalbuminuria; and 0.64 [0.490.82] for macroalbuminuria), but there was no statistically significant association between pro12ala polymorphism and esrd . Furthermore, in the analysis stratified by ethnicity, significant association was detected between the polymorphism and dn risk in caucasian (0.68 [0.510.90]) but not in asian populations (0.85 [0.621.17]). Significant heterogeneity was eliminated in most of the subgroup analyses but the asian groups (ph = 0.05; i = 48). The details are also listed in table 1 . To test the stability of the pooled results, one - way sensitivity analyses of the pooled ors and when omitting each dataset in the overall meta - analysis, the pooled ors were always persistent (fig . 2). Begg and egger tests were used to provide diagnosis of the potential publication bias, and no evidence of publication bias was found in the overall analysis (pbegg = 0.150; pegger = 0.085) (table 1 and fig . 3). Evidence also suggested no publication bias in the subanalyses mentioned above, but one for the association between ppar pro12ala and albuminuria (pbegg = 0.063; pegger = 0.036). However, after getting rid of one dataset each by caramori (24), de cosmo (7), pollex (21), or li (19), the existing bias disappeared (pbegg = 0.100, pegger = 0.053; pbegg = 0.100, pegger = 0.060; pbegg = 0.100, pegger = 0.089; and pbegg = 0.100, pegger = 0.056, respectively), whereas the significant ors were persistent . The persistent result indicated that the emerging publication bias did not decrease the reliability of the result . One - way sensitivity analysis of the pooled ors and 95% ci for the overall analysis, omitting each dataset in the meta - analysis . The se is plotted against a logarithmic scale of the or [log(or)]. We observed that levels of scr were significantly associated with the pro12ala polymorphism of ppar in t2 dm subjects (supplementary fig . 2, bottom). Overall, the smd for scr was statistically significant (smd = 0.12; 95% ci 0.23 to 0.01; z = 2.13, pz = 0.03). There was no significant heterogeneity among the trials (ph = 0.52; i = 0%). The effects of the polymorphism on the level of aer were all significant in the four included trials (supplementary fig . The synthetic smd for aer was significant (smd = 3.04; 95% ci 4.90 to 1.18; z = 3.20, pz = 0.001). However, the heterogeneity among eligible studies (ph <0.001; i = 99%) was significant . It is noteworthy that a significant decline in aer was detected in the individuals with 12ala, whereas no evidence of publication bias was observed (supplementary fig . Genetic epidemiologic studies of single nucleotide polymorphism, if large and unbiased, can provide evidence of the association between candidate gene and disease risk . This human genome epidemiology association review is an updated meta - analysis of the relationship between the ppar pro12ala polymorphism and susceptibility of dn in t2 dm patients . The overall meta - analysis, based on 18 case - control studies involving 3,361 case subjects and 5,825 control subjects, indicates that the polymorphism is associated with the risk of t2dm - dn . Considering the significant heterogeneity among the identified studies, subgroup analyses are also performed, stratified by ethnicity of population and stage of nephropathy . In selected subgroup of caucasians but not asians, we observe a significant association between ala allele and the risk of t2dm - dn . In the other subanalysis, we also observe that the pro12ala is associated with decreased albuminuria risk (i.e., microalbuminuria and macroalbuminuria), whereas there is no relationship between the polymorphism and esrd risk . So we also make two additional analyses to investigate the effect of the pro12ala polymorphism on the scr and aer . As expected, we do see an association of the pro12 allele with higher scr and aer levels . Ethnicity difference and stage of disease may be responsible for the significant heterogeneity among the studies included in the overall analysis, so subgroup analyses stratified by ethnicity and stage of dn are addressed . In the caucasian subgroup, the absence of a statistical effect in the asian subgroup might be explained by the low frequency of ala12 allele in asian populations, the distinction between various races varies from 4% in asian to 28% in caucasian populations (28). The much lower frequency of the ala carriers in the asian population requires a larger sample to detect statistically significant association . There may also be false positives in the caucasian subgroup due to the control subjects not having the same allele frequency as the cases because the cases and control subjects by chance come from different populations (the brazilians and turks are both mixed race, although they mostly consist of caucasians). Additionally, gene gene and gene environmental interactions should also contribute to the different results (29). The ethnic difference may be also induced by curative activities, such as drug use or age at the first diagnosis, although these have to be further confirmed by large population studies . The first reason that we fail to detect an association between the polymorphism and esrd risk in our patients with t2 dm might be related to the fact that esrd is more complex than albuminuria, which is determined by plenty of pathophysiological factors . In addition, patients with t2 dm usually die of other complications, such as cardiovascular risk, before developing esrd (30,31); therefore, no significant association between the polymorphism and esrd is introduced . Additionally, as only a few of the esrd subjects are available, the reliability of this result remains suspicious . Ppar has been implicated in almost all of the pathological processes contributing to atherosclerosis, including endothelial dysfunction, leukocyte chemotaxis, foam cell formation, and plaque evolution, destabilization, and rupture (32). However, the mechanisms by which the variations of ppar gene actually protect against dn remain incompletely understood . Various mouse models of ppar deficiency have been generated to dissect the function of ppar. The study of these models has confirmed that ppar plays a key role in regulating insulin sensitivity (33,34), which has been proved to be closely associated with glomerular filtration rate and albuminuria (35). The amp - activated protein kinase signaling pathway may play a critical role in mediating the insulin - sensitizing action of ppar (36). Other studies also identify that thiazolidinedione - induced adiponectin may sensitize the insulin action through the amp - activated protein kinase - dependent pathway in adipose tissue, skeletal muscle, and liver (37,38). Additionally, a recent study addressed by cabezas et al . (39) revealed that ppar and its agonists positively control megalin expression; this regulation could have an important impact on several megalin - mediated physiological processes and pathophysiologies such as chronic kidney disease associated with diabetes and hypertension . The stability of the meta - analysis was verified by one - way sensitivity analysis, which pooled ors by omitting each dataset included . It may indicate that the included patients effectively maintained the most important inherent nature of population in genetic structure and that largely improved the predictability and reliability of the meta - analysis . Moreover, to confirm the reliability of our results, we minimized the possible publication bias by performing searches comprehensively and designing this study precisely . Additionally, begg and egger tests were used to test the potential publication bias, and no significant evidence was observed in the overall analysis . However, some limitations of our study should be acknowledged . First, the number of esrd patients is relatively small for exploring the reliable result with enough statistical power in the subgroup analyses; second, our results are based on unadjusted estimates, whereas a more precise analysis should be conducted if other covariates (i.e., age, sex, and so on) are available . Despite these limitations, our meta - analyses suggest that the ppar 12ala allele significantly decreases the t2dm - dn risk and the level of scr and aer . More well - designed studies with larger sample sizes and more details on individuals are needed to provide more precise evidence to further confirm our meta - analysis.
Detailed descriptions of the two multicenter, randomized, double - blind, placebo - controlled phase 3 studies, including inclusion and exclusion criteria and vms efficacy endpoints, have been published previously.30 the treatment period was 12 weeks in the first study (clinicaltrials.gov identifier nct01361308) and 24 weeks in the second study (clinicaltrials.gov identifier nct01101841). Otherwise, the two studies were similar in screening, placebo run - in period, population enrolled, and treatment regimens . In brief, participants in both studies were postmenopausal women aged at least 40 years at screening who met one of the following criteria for menopause: spontaneous amenorrhea for 12 consecutive months or more; amenorrhea for 6 months or more, with follicle - stimulating hormone levels higher than 40 miu / ml; or bilateral salpingo - oophorectomy, with or without hysterectomy, 6 weeks or more before screening . A key inclusion criterion was an average of more than 7 to 8 moderate to severe hot flashes / day or 50 to 60 moderate to severe hot flashes / week reported for 30 days or more before screening . Psychotropic drugs, including all sedative and hypnotic medications (with the exception of zolpidem, zaleplon, eszopiclone, and diphenhydramine), were prohibited during the study . Use of nightly zolpidem, zaleplon, eszopiclone, and diphenhydramine was minimal, and no analysis was performed with respect to the use of these medications . Participants taking psychotropic drugs or estrogen / progestin - containing products were required to undergo prespecified washout periods before the run - in visit . Key exclusion criteria were as follows: known nonresponse of vms to previous ssri or serotonin - norepinephrine reuptake inhibitor treatment, untreated hypertension, impaired liver or kidney function, unstable cardiac disease, pregnancy, history of self - injurious behavior, history of clinical diagnosis or treatment of depression or any other psychiatric disorder (including substance abuse or alcohol disorders), and any other ongoing medical condition . No sleep - specific inclusion or exclusion criteria (such as presence of sleep apnea or restless legs) were applied . Each study began with a single - blind, placebo run - in period of up to 12 days, during which eligible participants received placebo once daily at bedtime and used electronic daily diaries to record the number and severity of vms and the number of nighttime awakenings that they attributed to vms . A double - blind treatment period followed, during which participants were randomly assigned 1:1 to receive paroxetine 7.5 mg or an identical capsule of placebo once daily at bedtime for 12 or 24 weeks . Randomization was applied centrally across all sites using an interactive voice response system, and all personnel were blinded to study medication until study completion and database lock . Participants recorded vms daily using a real - time interactive voice or web response system that was accessible 24 hours / day; primary vms efficacy endpoints have been reported elsewhere.30 sleep parameters included change from baseline in the total number of nighttime awakenings attributed to vms (where participants were asked to self - record all nighttime awakenings that they attributed to vms in the electronic sleep diary the following morning between 6 am and 11 am; appendix) and other sleep - related measurements (sleep - onset latency and hours of sleep per night; appendix). Data were recorded throughout the study period and were collected for analysis in both studies on day 1 (study start), day 28 (week 4), and day 85 (week 12; end of 12-wk study) and for the 24-week study on day 169 (week 24; end of 24-wk study). In addition, the extent to which vms interfered with sleep was measured on weeks 4, 12, and 24 using the sleep interference item from the 10-item validated hot flash related daily interference scale (hfrdis),40 with scores ranging from 0 (no interference with sleep associated with hot flashes) to 10 (interference with sleep associated with hot flashes to the worst possible extent). The proportion of women with moderate to severe difficulty sleeping was also assessed using the validated 21-item greene climacteric scale (gcs),41 which includes a question on difficulty in sleeping (0, none; 1, mild; 2, moderate; 3, severe). The discontinuation - emergent signs and symptoms scale, which includes the symptoms trouble sleeping / insomnia and increased dreaming or nightmares, was administered within a mean (sd) of 7 (3) days of the last dose of study medication, regardless of when the participant exited the study . The principal population for analysis of secondary endpoints was the modified intent - to - treat population (all consenting and randomly assigned participants with valid baseline daily vms diary data who had taken one or more doses of study medication and had one or more days of on - treatment daily vms diary data). The safety population comprised all participants who received one or more doses of study medication and who had one or more postdose safety measurements . In this prospective analysis of sleep - related predefined secondary endpoints, data from the two phase 3 studies were pooled for weeks 1 to 12 . The two studies were similarly designed and involved comparable populations, allowing pooling of data, which enables evaluation of information from more than 1,100 participants and makes the data set and analysis more robust . Prespecified analyses were used to assess the impact of paroxetine 7.5 mg on the number of nighttime awakenings attributed to vms, sleep - onset latency, sleep duration, and sleep scores (hfrdis and gcs). For each participant, total nighttime awakenings attributed to vms at baseline were calculated as the average during the run - in period before randomization . Nighttime awakenings during the double - blind treatment period were calculated as the average for each study week . Sleep - onset latency and duration of sleep were calculated as change from baseline in number of minutes at each postbaseline time point evaluated . Detailed descriptions of the two multicenter, randomized, double - blind, placebo - controlled phase 3 studies, including inclusion and exclusion criteria and vms efficacy endpoints, have been published previously.30 the treatment period was 12 weeks in the first study (clinicaltrials.gov identifier nct01361308) and 24 weeks in the second study (clinicaltrials.gov identifier nct01101841). Otherwise, the two studies were similar in screening, placebo run - in period, population enrolled, and treatment regimens . In brief, participants in both studies were postmenopausal women aged at least 40 years at screening who met one of the following criteria for menopause: spontaneous amenorrhea for 12 consecutive months or more; amenorrhea for 6 months or more, with follicle - stimulating hormone levels higher than 40 miu / ml; or bilateral salpingo - oophorectomy, with or without hysterectomy, 6 weeks or more before screening . A key inclusion criterion was an average of more than 7 to 8 moderate to severe hot flashes / day or 50 to 60 moderate to severe hot flashes / week reported for 30 days or more before screening . Psychotropic drugs, including all sedative and hypnotic medications (with the exception of zolpidem, zaleplon, eszopiclone, and diphenhydramine), were prohibited during the study . Use of nightly zolpidem, zaleplon, eszopiclone, and diphenhydramine was minimal, and no analysis was performed with respect to the use of these medications . Participants taking psychotropic drugs or estrogen / progestin - containing products were required to undergo prespecified washout periods before the run - in visit . Key exclusion criteria were as follows: known nonresponse of vms to previous ssri or serotonin - norepinephrine reuptake inhibitor treatment, untreated hypertension, impaired liver or kidney function, unstable cardiac disease, pregnancy, history of self - injurious behavior, history of clinical diagnosis or treatment of depression or any other psychiatric disorder (including substance abuse or alcohol disorders), and any other ongoing medical condition . No sleep - specific inclusion or exclusion criteria (such as presence of sleep apnea or restless legs) were applied . Each study began with a single - blind, placebo run - in period of up to 12 days, during which eligible participants received placebo once daily at bedtime and used electronic daily diaries to record the number and severity of vms and the number of nighttime awakenings that they attributed to vms . A double - blind treatment period followed, during which participants were randomly assigned 1:1 to receive paroxetine 7.5 mg or an identical capsule of placebo once daily at bedtime for 12 or 24 weeks . Randomization was applied centrally across all sites using an interactive voice response system, and all personnel were blinded to study medication until study completion and database lock . Participants recorded vms daily using a real - time interactive voice or web response system that was accessible 24 hours / day; primary vms efficacy endpoints have been reported elsewhere.30 sleep parameters included change from baseline in the total number of nighttime awakenings attributed to vms (where participants were asked to self - record all nighttime awakenings that they attributed to vms in the electronic sleep diary the following morning between 6 am and 11 am; appendix) and other sleep - related measurements (sleep - onset latency and hours of sleep per night; appendix). Data were recorded throughout the study period and were collected for analysis in both studies on day 1 (study start), day 28 (week 4), and day 85 (week 12; end of 12-wk study) and for the 24-week study on day 169 (week 24; end of 24-wk study). In addition, the extent to which vms interfered with sleep was measured on weeks 4, 12, and 24 using the sleep interference item from the 10-item validated hot flash related daily interference scale (hfrdis),40 with scores ranging from 0 (no interference with sleep associated with hot flashes) to 10 (interference with sleep associated with hot flashes to the worst possible extent). The proportion of women with moderate to severe difficulty sleeping was also assessed using the validated 21-item greene climacteric scale (gcs),41 which includes a question on difficulty in sleeping (0, none; 1, mild; 2, moderate; 3, severe). The discontinuation - emergent signs and symptoms scale, which includes the symptoms trouble sleeping / insomnia and increased dreaming or nightmares, was administered within a mean (sd) of 7 (3) days of the last dose of study medication, regardless of when the participant exited the study . The principal population for analysis of secondary endpoints was the modified intent - to - treat population (all consenting and randomly assigned participants with valid baseline daily vms diary data who had taken one or more doses of study medication and had one or more days of on - treatment daily vms diary data). The safety population comprised all participants who received one or more doses of study medication and who had one or more postdose safety measurements . In this prospective analysis of sleep - related predefined secondary endpoints, data from the two phase 3 studies the two studies were similarly designed and involved comparable populations, allowing pooling of data, which enables evaluation of information from more than 1,100 participants and makes the data set and analysis more robust . Prespecified analyses were used to assess the impact of paroxetine 7.5 mg on the number of nighttime awakenings attributed to vms, sleep - onset latency, sleep duration, and sleep scores (hfrdis and gcs). For each participant, total nighttime awakenings attributed to vms at baseline were calculated as the average during the run - in period before randomization . Nighttime awakenings during the double - blind treatment period were calculated as the average for each study week . Sleep - onset latency and duration of sleep were calculated as change from baseline in number of minutes at each postbaseline time point evaluated . In total, 614 participants were randomly assigned to the 12-week study, and 570 participants were randomly assigned to the 24-week study; overall, more than 80% of women completed the studies, and most were at least 80% compliant with study drug treatment.30 for the pooled analysis, the modified intent - to - treat population comprised 1,174 women (paroxetine 7.5 mg, n = 585; placebo, n = 589), and the safety population comprised 1,175 women (paroxetine 7.5 mg, n = 586; placebo, n = 589; fig . Demographic characteristics were not notably different between treatment arms at baseline, although statistical analyses were not performed on baseline demographic variables (table 1). At baseline, the mean (sd) daily frequency of vms (daytime plus nighttime) was 11.3 (4) in both groups.30 sleep - related characteristics at baseline were also similar between treatment arms: participants experienced a mean (sd) of 3.6 (2) nighttime awakenings attributed to vms (or 25 per week) and indicated that hot flashes interfered with sleep to a large extent, with an overall mean hfrdis score of 7.6 (table 1). Demographics and baseline characteristics (pooled data; mitt population) participant disposition (all randomly assigned populations). In the pooled analysis of data through week 12, the reduction in the number of nighttime awakenings attributed to vms was significantly greater among participants receiving paroxetine 7.5 mg than among participants receiving placebo on weeks 4 and 12 (fig . (pooled data), the reduction from baseline in nighttime awakenings attributed to vms was 39% (from 3.56 to 2.17) in the paroxetine 7.5 mg treatment arm compared with 28% (from 3.64 to 2.63) in the placebo arm (p = 0.0049). On week 12 (pooled data), reductions from baseline were 54% (from 3.56 to 1.65) in the paroxetine 7.5 mg arm and 43% (from 3.64 to 2.08) in the placebo arm (p = 0.0001). In the 24-week study, this effect was sustained for the entire treatment period, with participants in the paroxetine 7.5 mg arm experiencing a 62% reduction from baseline in nighttime awakenings attributed to vms (from 3.58 to 1.36) compared with 43% (from 3.56 to 2.02) in the placebo arm (p <0.0001). Mean change from baseline in daily nighttime awakenings attributed to vasomotor symptoms (a), duration of sleep (b), and sleep - onset latency (c). The duration of sleep per night increased significantly more among participants receiving paroxetine 7.5 mg than among those receiving placebo at all postbaseline time points (fig . Participants receiving paroxetine 7.5 mg had a significant increase in sleep time (+ 8%, 31 min) compared with those receiving placebo on week 4 (pooled data: + 4%, 16 min; p = 0.0075), week 12 (pooled data: paroxetine 7.5 mg, 9%, 35 min; placebo, 6%, 23 min; p = 0.0102), and week 24 (24-wk study: paroxetine 7.5 mg, 10%, 37 min; placebo, 7%, 27 min; p = 0.0336). No significant differences in sleep - onset latency were noted between the two treatment arms during the course of the study (fig . 2c). On week 4 (pooled data), sleep - onset latency was reduced by 14% (7 min) in participants receiving paroxetine 7.5 mg and by 15% (8 min) in those receiving placebo (p = 0.3509). On week 12 (pooled data), sleep - onset latency was reduced by 22% (11 min) and 27% (14 min; p = 0.4875), respectively . On week 24 (24-wk study), reductions were 30% (15 min) and 27% (12 min; p = 0.1966), respectively . 3a) were reduced from baseline to a significantly greater extent in the paroxetine 7.5 mg arm than in the placebo arm on week 4 (pooled data: paroxetine 7.5 mg, 32%; placebo, 20%; p = 0.0068), but not on week 12 (pooled data: paroxetine 7.5 mg, 42%; placebo, 35%; p = 0.0663) or on week 24 (24-wk study: paroxetine 7.5 mg, 51%; placebo, 38%; p = 0.2405). Mean change from baseline in sleep interference scores (a) and in the gcs sleep item difficulty in sleeping hfrdis, hot flash related daily interference scale; gcs, greene climacteric scale . On the gcs sleep item difficulty in sleeping, the proportions of participants reporting moderate to severe difficulty sleeping decreased from baseline in both treatment arms; however, the differences between treatment groups were not statistically significantly different at any time point (fig . The proportion of participants reporting a sleep - related teae was low in both treatment arms . During the treatment period, 3.1% of women in both treatment arms reported at least one sleep - related teae; after treatment discontinuation, 1.4% in the paroxetine 7.5 mg arm and 0.3% in the placebo arm reported a sleep - related teae . No statistically significant differences in individual sleep - related teaes were reported between treatment arms (table 2). Furthermore, according to the results of the discontinuation - emergent signs and symptoms scale, no meaningful differences in discontinuation - emergent signs and symptoms were noted between treatment arms after treatment discontinuation without tapering . Sleep - related adverse events reported during the double - blind treatment period and posttreatment period (pooled data; safety population) in total, 614 participants were randomly assigned to the 12-week study, and 570 participants were randomly assigned to the 24-week study; overall, more than 80% of women completed the studies, and most were at least 80% compliant with study drug treatment.30 for the pooled analysis, the modified intent - to - treat population comprised 1,174 women (paroxetine 7.5 mg, n = 585; placebo, n = 589), and the safety population comprised 1,175 women (paroxetine 7.5 mg, n = 586; placebo, n = 589; fig . Demographic characteristics were not notably different between treatment arms at baseline, although statistical analyses were not performed on baseline demographic variables (table 1). At baseline, the mean (sd) daily frequency of vms (daytime plus nighttime) was 11.3 (4) in both groups.30 sleep - related characteristics at baseline were also similar between treatment arms: participants experienced a mean (sd) of 3.6 (2) nighttime awakenings attributed to vms (or 25 per week) and indicated that hot flashes interfered with sleep to a large extent, with an overall mean hfrdis score of 7.6 (table 1). Demographics and baseline characteristics (pooled data; mitt population) participant disposition (all randomly assigned populations). In the pooled analysis of data through week 12, the reduction in the number of nighttime awakenings attributed to vms was significantly greater among participants receiving paroxetine 7.5 mg than among participants receiving placebo on weeks 4 and 12 (fig . (pooled data), the reduction from baseline in nighttime awakenings attributed to vms was 39% (from 3.56 to 2.17) in the paroxetine 7.5 mg treatment arm compared with 28% (from 3.64 to 2.63) in the placebo arm (p = 0.0049). On week 12 (pooled data), reductions from baseline were 54% (from 3.56 to 1.65) in the paroxetine 7.5 mg arm and 43% (from 3.64 to 2.08) in the placebo arm (p = 0.0001). In the 24-week study, this effect was sustained for the entire treatment period, with participants in the paroxetine 7.5 mg arm experiencing a 62% reduction from baseline in nighttime awakenings attributed to vms (from 3.58 to 1.36) compared with 43% (from 3.56 to 2.02) in the placebo arm (p <0.0001). Mean change from baseline in daily nighttime awakenings attributed to vasomotor symptoms (a), duration of sleep (b), and sleep - onset latency (c). The duration of sleep per night increased significantly more among participants receiving paroxetine 7.5 mg than among those receiving placebo at all postbaseline time points (fig . Participants receiving paroxetine 7.5 mg had a significant increase in sleep time (+ 8%, 31 min) compared with those receiving placebo on week 4 (pooled data: + 4%, 16 min; p = 0.0075), week 12 (pooled data: paroxetine 7.5 mg, 9%, 35 min; placebo, 6%, 23 min; p = 0.0102), and week 24 (24-wk study: paroxetine 7.5 mg, 10%, 37 min; placebo, 7%, 27 min; p = 0.0336). No significant differences in sleep - onset latency were noted between the two treatment arms during the course of the study (fig . (pooled data), sleep - onset latency was reduced by 14% (7 min) in participants receiving paroxetine 7.5 mg and by 15% (8 min) in those receiving placebo (p = 0.3509). On week 12 (pooled data), sleep - onset latency was reduced by 22% (11 min) and 27% (14 min; p = 0.4875), respectively . On week 24 (24-wk study), reductions were 30% (15 min) and 27% (12 min; p = 0.1966), respectively . 3a) were reduced from baseline to a significantly greater extent in the paroxetine 7.5 mg arm than in the placebo arm on week 4 (pooled data: paroxetine 7.5 mg, 32%; placebo, 20%; p = 0.0068), but not on week 12 (pooled data: paroxetine 7.5 mg, 42%; placebo, 35%; p = 0.0663) or on week 24 (24-wk study: paroxetine 7.5 mg, 51%; placebo, 38%; p = 0.2405). Mean change from baseline in sleep interference scores (a) and in the gcs sleep item difficulty in sleeping hfrdis, hot flash related daily interference scale; gcs, greene climacteric scale . On the gcs sleep item difficulty in sleeping, the proportions of participants reporting moderate to severe difficulty sleeping decreased from baseline in both treatment arms; however, the differences between treatment groups were not statistically significantly different at any time point (fig . The proportion of participants reporting a sleep - related teae was low in both treatment arms . During the treatment period, 3.1% of women in both treatment arms reported at least one sleep - related teae; after treatment discontinuation, 1.4% in the paroxetine 7.5 mg arm and 0.3% in the placebo arm reported a sleep - related teae . No statistically significant differences in individual sleep - related teaes were reported between treatment arms (table 2). Furthermore, according to the results of the discontinuation - emergent signs and symptoms scale, no meaningful differences in discontinuation - emergent signs and symptoms were noted between treatment arms after treatment discontinuation without tapering . Sleep - related adverse events reported during the double - blind treatment period and posttreatment period (pooled data; safety population) in this pooled analysis of data from two phase 3 studies, treatment with paroxetine 7.5 mg significantly and sustainably reduced the mean number of nighttime awakenings attributed to vms and increased the duration of sleep per night compared with placebo without differentially affecting sleep - onset latency or ae reporting of sedation . Although interference of vms with sleep on the hfrdis was initially improved to a greater extent with paroxetine 7.5 mg than with placebo, this effect was not sustained . Taken together, these results suggest that paroxetine 7.5 mg had a selective effect on sleep parameters related to vms rather than a nonspecific effect on sleep parameters (ie, increasing sedation or inducing somnolence). Paroxetine 7.5 mg was specifically developed to treat moderate to severe vms associated with menopause.30 ssris such as paroxetine at doses prescribed for psychiatric conditions may adversely affect sleep37,42,43; therefore, we examined the effect of this low - dose formulation in a population of women without mental health issues . In this analysis of phase 3 data, treatment with paroxetine 7.5 mg significantly reduced the mean nighttime number of nighttime awakenings attributed to vms within 1 month of the start of therapy (1.39) compared with placebo (1.01; a difference of 0.38 additional awakenings reduced by paroxetine vs placebo). Furthermore, the magnitude of the reduction was sustained, and even increased, after 12 and 24 weeks of ongoing paroxetine 7.5 mg treatment . Although the overall reduction of 0.38 in nighttime awakenings is only a moderate effect, it may be clinically meaningful for some women, potentially resulting in positive benefits for daily functioning and quality of life . Of note, only the number of nighttime awakenings associated with vms was recorded in these phase 3 studies; there is no available information on whether the number of awakenings unrelated to vms was altered or whether the duration of nighttime awakenings attributed to vms changed . In addition to reducing the number of nighttime awakenings attributed to vms, paroxetine 7.5 mg significantly increased the duration of sleep per night, indicating a link between reduced nighttime awakenings and improvement in overall sleep duration . The improvement in sleep duration with paroxetine 7.5 mg (an increase of up to 37 min / night from baseline) compares favorably with results obtained with agents used specifically for the treatment of insomnia, such as zolpidem, which has been shown to increase total sleep time by 51 minutes or more per night.44,45 studies using objective sleep assessments have shown that nighttime vms do not extensively alter sleep architecture,13 suggesting that reducing vms may be sufficient to restore normal sleep patterns . Additional studies would allow a direct correlation of sleep improvements with changes in menopause - specific quality - of - life measures . No significant differences in sleep - onset latency were observed between paroxetine 7.5 mg and placebo . It is notable that baseline levels of sleep - onset latency were higher than expected for a group of women not selected for sleep problems or insomnia . Sleep modeling studies may provide further information on whether sleep effects attributable to paroxetine 7.5 mg treatment are direct or indirect and may define how sleep duration is increased without differential reduction in sleep - onset latency . The use of the hfrdis in the present analysis to assess the impact of vms on sleep indicates that, during the double - blind treatment period, scores in both treatment arms were reduced within 4 weeks of treatment initiation (pooled data: paroxetine 7.5 mg, 2.41; placebo, 1.50) and reductions continued for up to 24 weeks of treatment (24-wk study: paroxetine 7.5 mg, 3.84; placebo, 2.89). Although between - treatment group differences were significant only on week 4, a trend toward greater improvement with paroxetine 7.5 mg was observed on weeks 12 and 24 . However, it must be noted that use of a single item from the hfrdis to evaluate sleep has not been validated . The proportion of participants with moderate to severe difficulty sleeping, according to the gcs, decreased in both groups at all time points, but differences between treatment arms were not statistically significantly different . Future studies would benefit from the inclusion of specific sleep interference scales, such as the insomnia severity index (isi),46 to identify the importance of sleep improvement for participants . The frequency of sleep - related aes, such as somnolence, at doses of paroxetine prescribed for psychiatric conditions is dose - related,42 suggesting that lower doses (such as 7.5 mg) may result in fewer such aes . Consistent with this hypothesis, somnolence was reported as an ae in only 0.5% of paroxetine 7.5 mg treated women in the paroxetine 7.5 mg phase 3 studies, further reinforcing the supposition that the effects of paroxetine 7.5 mg on sleep are probably a result of a reduction in vms rather than sedation . In a phase 2 trial of paroxetine 7.5 mg, sleep parameters were not assessed, but neither insomnia nor somnolence was reported as an ae.47 previous publications have observed a link between vms associated with menopause and increased prevalence of sleep disturbances and nighttime awakenings.9 a subcohort single - site study of 51 white and african - american postmenopausal women who participated in the study of women s health across the nation evaluated whether vms during sleep were associated with poorer sleep . Measures included sternal skin conductance to capture vms, actigraphy to objectively assess sleep, a vms diary, and a sleep diary completed before sleep and on awakening.10 for these women, vms reported on awakening but not physiologically assessed vms or vms reported during sleep were related to poorer actigraphic sleep.10 a single - cohort university study (do stage transitions result in detectable effects [stride]) evaluated associations among sleep disturbance and the frequency, bothersomeness, and interference of hot flashes in 623 women at various menopause stages . Here, vms were assessed annually during a 2-week period using the hfrdis, and women self - reported on sleep . In multivariable models, women reporting bothersome hot flashes were more likely to report sleep disturbances than women who reported no vms (odds ratio, 2.1; 95% ci, 1.4 - 3.2).11 in another recent study, estradiol was suppressed by leuprolide in 29 healthy premenopausal volunteers, and vms were assessed using polysomnography, a sleep diary, and standardized questionnaires.13 increasing nighttime vms frequency measured by polysomnography resulted in increased wake after sleep onset time and number of awakenings, validating the subjective experiences of women reporting awakenings in the sleep diary.13 the isi46 and the pittsburgh sleep quality index (psqi)48 were used to evaluate subjective sleep quality in a recent 8-week randomized study of the ssri escitalopram (10 - 20 mg / d) versus placebo in 205 perimenopausal / postmenopausal women with vms.36 treatment with escitalopram significantly reduced isi and psqi scores on week 8 compared with placebo (p <0.001 for both). Clinical improvements in insomnia symptoms and subjective sleep quality (defined as 50% decreases in the isi and psqi from baseline) occurred more frequently in escitalopram - treated women than in the placebo group.36 although these data support the inference from paroxetine 7.5 mg studies that ssri treatment can improve sleep in postmenopausal women with vms, a direct comparison of outcomes is not possible because different sleep measures were used in each study . Hormone therapy is beneficial for treating vms associated with menopause16,17 and improves sleep disturbances related to vms in perimenopausal and early postmenopausal women.1,4,18 in a randomized cross - over study of 63 postmenopausal women, hormone therapy demonstrated significantly improved sleep quality and fewer nighttime awakenings compared with placebo (p <0.001).49 furthermore, women treated with hormone therapy experienced a longer duration of rapid eye movement sleep, shortened sleep latency, and improvement in sleep efficiency.50 however, nonhormonal treatments are needed for women for whom hormone therapy is contraindicated and for those unwilling to be treated with hormone therapy . The major strength of the present analysis is the prospective randomized gathering of sleep information from a large number of study participants, with no exclusions for sleep apnea or restless legs (the most common diagnoses interrupting sleep beyond vms); thus, the findings are generalizable to the larger population . Use of data from two similarly designed studies with similar patient populations allowed pooling of data and comparison with baseline values, providing a more robust evaluation of outcomes . An additional strength of this analysis is the inclusion of minority and overweight study participants (table 1). The fact that the instruments used for vms and sleep analysis were self - reported, rather than objectively measured, is a limitation of this analysis; however, self - perception of vms bothersomeness and sleep disruption is of particular clinical relevance . That sleep outcomes were secondary, rather than primary, endpoints is another potential limitation of this analysis in the phase 3 studies, paroxetine 7.5 mg once daily for the treatment of vms in postmenopausal symptomatic women significantly reduces the number of nighttime awakenings attributed to vms and increases the duration of sleep . Although the overall reduction in nighttime awakenings is moderate, it has been sustained through 12 and 24 weeks of treatment with paroxetine 7.5 mg and may represent a meaningful magnitude of improvement for some women with symptoms attributed to vms . Improvements in nighttime awakenings and sleep duration occur without increased sedation, reduced sleep - onset latency, or increased sleep - related aes, suggesting that, in these studies, paroxetine 7.5 mg has a selective therapeutic effect on sleep parameters related to vms.
The energy level of its lowest lying degenerate vibration is only e = 171 cm, consequently according to the boltzman distribution law it has a population of 58% (29%2 for vibrational degeneracy) of the ground state at dry ice temperature (200 k). Therefore the rotational spectrum in the v10 = 1 state is also strong and it is not complicated by interactions with nearby vibrational states . Whereas it is a prolate top, its rotational constants a 5.7 and b 2.9 ghz are not large . So, it is a possible candidate for the observation of the a1-a2 splitting in the ground state . The qt+ is rather large and a b a is small and it is possible to observe direct -type resonance transitions in the microwave range . These situation have been previously observed only for pf3, v4 = 1, for cf3h and cf3d, v6 = 1, cf3cl, v6 = 1 and ch3cf3, v12 = 1 . The rotational spectra of cf3ccd, the isotopomer of this molecule in the ground, v10 = 1 and v10 = 2 states have been measured and studied by several authors [711]. Three identical fluorine atoms (i = 1/2) in cf3cch have important consequences in the determination of relative intensities of signals . The symmetry operations like exchange of nuclei, have symmetric effect on the total wavefunction for nuclei with integral or zero spin (bose particles). But for nuclei with half integral spin (fermi particles) the total wavefunction is antisymmetric . The rotation about the c3 axis through 2/3 or 4/3 rotation is equal to two exchanges of nuclei . Therefore it is symmetric (a) for fermi particles as well as bose particles . A rotation of 2/3 about top axis will not change rot states with k = 3n (n = integer, including n = 0) since rot e. for the symmetric state, the species are a and in antisymmetric state (k 3n) the species are e. there are eight different possibilities for combinations of three fluorine atoms with spin 1/2 . Two of them can occur when three spins are the same 1/2 or 1/2, these are totally symmetric because they remain unchanged by any permutation of nuclei which are equivalent to a rotation . But this situation cannot take place for the remaining 6 arrangements, which are 2a1 + 2e . The statistical weight depends on the ratio between the spin function of symmetry a and the spin function of symmetry e. consequently: for symmetric top molecules like cf3cch, the ground vibrational state rotational energies are given up to sextic terms by equation (1) and by application of selection rules j = + 1 and k = 0, the frequencies are given by equation (2). The spectra in this state are simple, so the different k values (k = 0,1,2,3,4 ...) for each j transition are assigned easily . This is illustrated in figure 1, which shows part of the observed transition j = 24 25, and compares it with the calculated spectrum . The centrifugal distortion produces a band head to high frequency at k = 0, with a spread to lower frequency with higher k figure 2 . Due to negative value of djk for ch3cl, this situation is different figure 3 . If \|k\| values are plotted against frequency for this state, the fortrat diagrams are produced which are shown in figures 2,3 . In these diagrams the splitting increase as k increases and this is due to the djk parameter . Djk is 6.27 and 2.517 khz for cf3cch and chcl3 respectively [2, 12]. The sextic centrifugal distortion constants were accurately determined for the first time for the ground state, and result shows that hjk> \|hkj\| and hkj <0 . For this molecule have found amount of this value, which is h3=2.310 - 21b4a - b . The predicted value of h3 for cf3cch, 2.61011 mhz, gives a splitting about 12 mhz for the highest - j transition (j = 112). Because v10 state of this molecule has a high population, the rotational spectra in this state are also strong . It is isolated in frequency from other vibrational states, so is not complicated by interactions with nearby states . Rotational frequencies for transitions j j + 1 in the excited degenerate vibrational state vt = 1, t = 1 of molecules with axial symmetry were calculated by nielsen . Although the nielsen theory was fairly satisfactory for the rotation spectrum of these type of molecules, some of the calculated frequencies by this method were different from the observed frequencies . This formula was extended to the case of higher j value by gordy et al . . They also interpreted the transitions j = 4 5 and j = 8 9 of cf3cch in the excited state v10 = 1 . Then the frequencies of lines for transition j j + 1 were investigated and calculated for vt = 1 and vt = 2 states [15, 16]. The frequency of transition j j + 1 for molecules belonging to the point group c3v in singly excited vibrational state vt = 1, is given by the approximate perturbation expression, equation (3). Where has the value qt (j + 1) if (k 1) = 0 (- type doubling) or where is the -type doubling constant, b and a are rotational constants, and is the z - coriolis constant for the vibrational state vt . The degree of -resonance thus largely depends on the ratio of qt+ to the resonance denominator (ba + a). With low to moderate values of this denominator, the spectrum usually found consists of two extreme outer lines for (k-1) = 0 (the - doublets) and a center group of lines for (k-1) 0 whose structure depends very strongly on other parameters such as the centrifugal distortion constants . As the strength of the resonance increase, the lines of the center group spread out until the low \|k-1\| transitions approach the positions of the -doublets . So for large qt+ and small (a b a = 458 mhz) value it is possible to observe direct -resonance transitions in the microwave range . This is a typical pattern for c3v doubly degenerate states and is predominantly due to the combinations of the splitting of the positive and negative (k-1) series by - type resonance [which is inversely proportional to (k-1)] and the shift to low frequency due to djk [which is proportional to (k-1)]. Thus one series goes from high frequency at low k to low frequency at high k, while the other is at low frequency both for high and low k, with a head at some intermediate value . In order to obtain more accuracy than can be obtained from perturbation theory in rotational energies for singly excited vibrational states it is necessary to set up the rotational hamiltonian as a matrix (h) in equation h = e and diagonalise to obtain the energy (2,10,11,17,18,21,22). This is particularly necessary when[b a + (a)] is small in the above equation; the diagonalization avoids the errors implicit in perturbation theory or the need to go to higher orders of perturbation . The hamiltonian is set up for a symmetric top molecule like cf3cch [2, 11]. This rotationvibrational hamiltonian has two different blocks belong to the different = + 1 and = 1 series . K and are no longer good quantum numbers but (k -) or (k - 1) may be used to distinguish between the symmetry species; those levels with (k - 1) = 3n, where n is an integer, are of species a1 or a2 . If (k- 1) 3n the species are e. the qt produces a first order splitting of the (k - 1) = 0, a1a2 pair which are the familiar - doublets, as shown by grenier - besson and amat and others [2, 10, 11, 15, 19]. The main difference from the ground state spectra is the splitting of the \|k - \| = 0 into two widely separate - doublets and the splitting, due to - resonance . Where is the vibration angular momentum quantum number and is the coriolis coupling coefficient . Hence k and are not good quantum numbers, though k - 1 may be used as a satisfactory label . The diagonal elements of the hamiltonian used were: in addition there are off - diagonal elements given by: in equation 5, the dj, djk, dk are quartic, hj, hjk, hkj are sextic centrifugal distortion terms respectively . Is the coriolis coupling coefficient between the two components of the degenerate state and j represents the centrifugal distortion of (a). The general effect of the additional parameters to complicate the spectrum relative to that of a non - degenerate state . If \|k 1\| values are plotted against frequency for this state, the fortrat - diagram is produced which is shown for ch3cch in figure 4 . This diagram shows the' ' doublet and two different series = 1 . So the spectrum is more complex compared with ground state . In the present example the rotational hamiltonian was diagonalised to give exact -resonance shifts . The spectrum in the millimetre - wave should consist of two widely spaced lines, the -doublets, whose separation is given by equation (4), and a central group of transitions with \|k 1\|> 0, whose frequencies are determined by equation (3). In the latter set the transitions with low values of \|k 1\| will tend to lie towards the outsides of the group since their -resonance shifts as predicted by equation (3) will be larges . There are two different series in spectrum, making it more difficult to assign than the ground state figure 4 . The vibrational levels closest to v10 = 2 are v5 = 1 and v9 = 1 at 536 and 452 cm, respectively [2, 9, 2022]. As the analysis will show, the level can be considered as being isolated and an effective hamiltonian for a vt = 2 level is suitable for the analysis of the data . The general features of the direct -resonance transitions in the vt = 2 level have been discussed by harder et al . . There is a good agreement for the obtained rotational constants b and the -dependence = (b2 b0)/4 . Because the main information is obtained from the data of carpenter et al . Excellent agreement is obtained for the values of g=ev2-ev0/4, because the parameter a constrained in its value . The b and other spectroscopic constants determined accurately up to v10 = 4 and show a regular dependence on the vibrational quantum number figure 5[2, 20, 22, 23]. These spectra show no obvious regularity from the point of view of frequencies as well as intensities, because there are three series for each j since the vibrational angular momentum quantum number can have values 0 and 2 . Therefore it is difficult to identify the quantum numbers k and associated with each absorption in these spectra . However from the point of view of experimental work, correspondence between the lines of different j value should be observed with respect to their relative position as well as their intensities . In order to obtain more accuracy in rotational energies for excited vibrational state vt = 2 it is necessary to set up the rotational hamiltonian as a matrix (h) in eigenvalue equation, h = e, and diagonalise to obtain the energy [2022]. This hamiltonian can set up for a symmetric top molecule such as cf3cch, cf3ccd, cf3cn, and opf3[10, 2030]. It has three different vibrational blocks related to = 2, 0 and 2 figure 6 . The vibrational anharmonicity x separates the = 2 and = 0 blocks and they are coupled by the matrix elements qt and rt . In order to explain the form of the spectra we need to consider the energy levels in vt = 2 as given by the hamiltonian matrix . The diagonal elements are: where 0 = pure vibrational frequency which is independent of j, k, and x is the anharmonicity factor in . The main off - diagonal elements are the -doubling elements equation (6). Plotting of 2(j+1) against (j + 1) can help us to analysis of signals in excited states . Which is described below? From the theoretical point the frequency expression for transition j j + 1 may be put in following effective form: where a(j, k,) and b(j, k,) are complicated functions of j, k and . For analysis and investigation of signals, equation (7) can be changed after some abbreviation and alteration to the following form: if both sides of eq (13) are divided by 2(j + 1), then: hence plotting 2(j+1) as a function of (j + 1) will produce a series of lines; two of them relate to k - 1 = 0 with slope - 2dj and intercept b - djkk212qt+ . The k - 1 0 states will produce a series of lines with slope and intercept b - djkk . Some of them are nearly parallel to each other and some of them show curvature due to strong resonance . Equation (10) can be further simplified by taking the term in dj to the right hand side . This is then an effective b value for the given transition: hence termed bplot . Plotting of beff as a function of (j + 1) will produce a series of lines, each of which belongs to a different (k - 1) value . Correlation of observed lines in different j can then be used to help with the assignment . In vt = 2 state the hamiltonian blocks off into factors which for a c3v molecule are of symmetry species a1, a2 and e. k and are no longer good quantum numbers but (k -) s remains a good symmetry label . In this label s is sign of, s = + 1 for 0 and s = 1 for <0 . The spectra are labled by (k-) such as 0, -1 and 0 in the spectrum of j = 19 figure 7 . Accidental resonances can occur when the = 0 series are coupled via the qt term with = 2 series . This resonance will affect the frequencies of the \|j, k,> \| j + 1, k,> transitions . The maximum resonance will occur when the energy of state = 2, k + 2 equals that of = 0, k i.e. After omitting terms which are the same from both sides of equation (8) this gives: if the rotational constants of cf3cch from table 1 are put in equation (18) then k is found to be 9.7 . Experimentally the resonance is indeed observed between k = 9 and k = 10 for this molecule and is independent of j, as expected from equation (18). The calculated spectrum of cf3cch in j = 20 21 show this resonance figure 8 . Like this resonance take places in different k values for different molecules which depends on their parameters table 2 . Figure 9 shows the energy levels as = 2, 0 and 2 stacks . The lines between states in adjacent stacks represent the -type interaction; for example k = 4 in = 2 is linked to k = 6, = 0 and this also interacts with k = (6 + 2) = 8, = + 2 . The interaction results in the energy levels being pushed apart; for example k = 4 is shifted up and k = 6 is shifted down . The extent of this shift depends on how nearly equal are the energy levels . When they are close, a large shift is obtained . The maximum shift will occur when the energies are almost equal, see equation (18) and is found in the region of k 9 . The hamiltonian for v10 = 3 has four vibrational blocks correspond to the = + 3, + 1, 1, 3 figure 10 . The = 3 and = 1 blocks are separated in vibrational energy by the anharmonic term . There are, however, couplings between + 3 and + 1, + 1, 1 and 1 and 3, due to the off - diagonal matrix elements qt and rt (abbreviated as q and r, respectively, in figures . 6, 10). The labelling of the eigenvalues of this hamiltonian by (k-) s where s is 1 for = + 3 and + 1, and 1 for = 3 and 1; the symmetry of a given transition can then be easily deduced from the fact that the extension to the v10 = 4 is straightforward . The possible values are + 4,+ 2, 0, 2, and 4, so that hamiltonian consists of five blocks and five series are apparent in the spectra . It is worth noting that, to the order of magnitude that was included in the hamiltonian . For v10 = 2, there are no new parameters in either of the above extension of this hamiltonian to the cases of v10 = 3 and v10 = 4 . For the cf3cch some of the lines belonging to the = 0, = 2 and = 2 are shown separately in different plots, (figures 11, 12, 13). In the = 2 series, the lines are nearly parallel to each other, and the distances between these lines become less as \|k - \|*s becomes smaller . The two lines 0 and 0, which are expected to have the same frequency but observed to be are separated, the line 0 is parallel with (j + 1) axis whereas 0 line is not figure 14 . The = 0 series are located between line 9 to line 10 but most of them are on the lower frequency side of the spectrum figure 11 . The lines 1 to 9 are above the 0 line whereas the other observed lines related to 10 and greater are hidden under the 0 lines figures . Line 10 is above line 0 and lines 11,12,13,14 cross line 0 but other observed lines belong to the = 0 are under the 0 line figures . 11,13 . Comparing of fortrat diagrams of v10 = 3 and v10 = 4 with corresponding for v10 = 1 figure . The series of v10 = 3 are qualitatively similar to those of v10 = 1, with -doublets and strong -resonance . In the same way the = 0 and + 2 series of v10 = 4 show a similar resonance to the corresponding series in v10 = 2 while the = 2 series is relatively unperturbed . In the case of v10 = 2, as k increase, the = 0 energies come into accidental resonance with those of the positive (k -) s series . Such a resonance is also present for v10 = 3, but in this case, because the vibrational separation due to the x term is so much larger, this resonance only starts to become obvious at high k . The maximum resonance can occur when omitting all centrifugal distortion and higher terms and terms which are the same for both states this gives: equation (20). If appropriate values input in equation 20 then solving this equation yields a value of k between 19 and 20 . There is a slight kink in this diagram which is caused by the accidental degeneracy of the \|j, k, = 1 and \|j, k + 4, = + 3 states which are not directly connected by a matrix element but are coupled in second order by the interaction of both of them with \|j, k = + 2, = + 1. applying the equation 21 yields a value for the k = 8.4 which is indeed where the perturbation is observed . The = 0 and = 2 series have separation in v10 = 4 which are close found in the v10 = 2 vibrational state . Consequently, this resonance arises in the same position found for v10 = 2, namely between k = 9 and 10 . The = 4 series are further away in the energy due to the x term . In the v10 = 4 spectrum figure . 15, the transition with (k -) s = 3 for the 2 series is split by about 3 mhz in the j = 20 spectrum . This splitting is not reproduced by the parameters obtained by extrapolation of the v10 = 2 and v10 = 3 values . In order to account for this, 4. this provides a first - order coupling between the otherwise degenerate energies of the k = + 1, = 2, and k = 1, = + 2 states which comprise the 3 pair of levels and leads to a splitting of this transition.
Age, gender, oral hygiene practices, and smoking are among the important risk factors that can affect the initiation, progression, and severity of periodontitis . The prevalence and severity of periodontal disease is found to increase with age . Furthermore, lack of oral hygiene encourages plaque formation, which leads to increase in pathogenic bacteria that are associated with severe forms of periodontal disease . Since time immemorial, tooth brushing has been considered ideal for maintaining a good oral hygiene . Despite the relationship between tooth brushing and periodontal disease being unclear; the breakthrough came in with rajala et al . Reporting the disease to be more prevalent in sporadic toothbrush users than otherwise . Additionally, a positive association between tooth brushing and low periodontal disease prevalence has been confirmed in some studies . Studies suggest that the smokers are 11 times more likely than non - smokers to harbor the bacteria that cause periodontal disease and are 4 times more likely to have advanced periodontitis . The sebha city is situated in south - western libya with a population of about 1,30,000 and people come from various urban areas surrounding it; therefore, it has a heterogeneous society with a low level of health education . As per leous periodontal disease is one of the main problems in libya; bleeding, dental calculus and shallow pockets is prevalent in almost all the young adults . It is also worth while to note that some of the 18-year - old male never clean their teeth with a toothbrush . The periodontal disease data from several countries collected by using cpitn, is stored in the who global oral health data bank . The sebha city belongs to the countries of the regional office for eastern mediterranean (emro). The oral health data for emro countries is available for the ages; 15 years, 17 years, 15 - 19 years, 16 - 20 years, 35 - 44 years, and 65 - 44 years . Usually, epidemiological studies are primarily concentrated on adolescents, adults, and elderly population . Unfortunately, no information is available for the young adults (18 - 34 years). The plausible reason for this lack of information comes from the fact that early studies presented results which suggested little or no periodontal destruction before the age of 30 years . Taking into consideration the lack of data about periodontal disease among young adults, the present study has been designed to assess the periodontal status among the young adults of sebha city . The study also assesses the relationship of the risk factors, such as age, gender, oral hygiene practices, and smoking with periodontal disease . A cross - sectional survey was conducted among the young adults aged 18 - 34 years from sebha city, libya . The city was divided into four zones, and one representative area from each zone was selected . Al - jadeed, al - mahdia, gurda, and sukkara were the four selected areas . This was carried out to obtain the prevalence of the periodontal disease so as to calculate the sample size for the main study . The pilot study results revealed that the prevalence of periodontal disease was 56% in al - jadeed . The sample size was determined by using the formula n = z pq / d . Assuming that the values obtained are z = a point on normal distribution with 95% confidence, p = prevalence of periodontal disease from pilot study, q = 100-p, d = admissible error that is 10% of prevalence . The survey was conducted at the primary health center (phc) in the selected areas . The medical officer posted at the phc was responsible for enrolling the participants for the study in the respective areas . Young adults who visited the phc for the 1 time and voluntarily wished to participate were recruited for the survey . Schedules were prepared such that the examination of the participant took place on one particular day of the week for one phc center . At first, the participant was interviewed to collect information as per the pro - forma [appendix 1] and later underwent a clinical examination . The oral examination was conducted by a single, trained, and calibrated examiner who was assisted by a recording clerk . 10% of the sample was re - examined and a kappa value of 0.8 was obtained . The codes and criteria of the index were as per the who, oral health survey . Three indicators of periodontal status; gingival bleeding, calculus, and pockets were assessed ., 10 index teeth were examined, which were (16, 17), 11, (26, 27), (36, 37), 31, and (46, 47). When two index teeth were considered in a sextant, the tooth with the highest score was recorded . For those below 20 years, only 6 index teeth were examined, which were 16, 11, 26, 36, 31, and 46 . The overall cpi score of the participant represented the value of the highest recorded code for that individual . During examination, the probe was kept parallel to the long axis of the tooth, the tip was inserted gently into the gingival sulcus or pocket, and the total extent of sulcus or pocket was explored . The codes were: code 0: teeth are healthycode 1: bleeding observed, directly or by using a mouth mirror, after probingcode 2: calculus detected during probing, all the black band on the probe is visiblecode 3: pocket is 4 - 5 mm (gingival margin within the black band on the probe)code 4: pocket is 6 mm or more (black band on the probe not visible)x: sextant is excluded (less than 2 teeth present)9: not recorded . Code 0: teeth are healthy code 1: bleeding observed, directly or by using a mouth mirror, after probing code 2: calculus detected during probing, all the black band on the probe is visible code 3: pocket is 4 - 5 mm (gingival margin within the black band on the probe) code 4: pocket is 6 mm or more (black band on the probe not visible) x: sextant is excluded (less than 2 teeth present) all the subjects were well - educated and signed the informed consent form . The ethical board, scientific committee of the faculty of dentistry, sebha university, sebha, libya approved the protocol of the study . Statistical analysis was carried out with the help of a statistical package for social sciences (spss version 16). Chi - square test and analysis of variance (anova) was used to compare between the groups at 5% level of significance . The variables were age, gender, frequency of tooth brushing, and smoking habit that were compared with the cpi codes . Out of the determined sample size of 1,256 people, the study population comprised of 1,255 individuals, of which 1,006 (80.15%) were females and 249 (19.84%) were males . The distribution of the sample as per the age and age groups is shown in table 1 . Distribution of the study sample according to the age regarding the oral hygiene practices, overall, 1,118 (89.08%) people reported to have used toothbrush and toothpaste for cleaning and 137 (10.91%) people used other aids, such as finger and siwak . Only 50 (3.98%) people were current smokers and all were males, of which maximum (n = 22) people smoked 10 - 20 cigarettes / day . Overall, the cpi analysis showed that a majority of 44.30% (n = 556) were detected with calculus (cpi code 2) followed by 40.63% (n = 510) with shallow pockets (cpi code 3), 6.29% (n = 79) with bleeding (cpi code 1), and 4.06% (n = 51) with deep pockets (cpi code 4). Only 4.7% (n = 59) had healthy periodontium (cpi code 0). Table 2 shows the distribution of people according to the cpi codes in relation to the different variables considered in the study . The frequency of tooth brushing and smoking were not significantly related to the cpi codes . Distribution of people according to the community periodontal index cpi codes in relation to the different variables the overall mean cpi score was 2.33 (0.84). Mean cpi score of the sample for different ages table 3 shows the mean (sd) number of sextants as per the cpi codes in relation to age . A maximum of 2.45 sextants were healthy among 18 years old, which further gradually decreased as the age advanced . From 19 years onward mean (sd) number of sextants as per the cpi codes in relation to age the literature search for information regarding the oral health status of the population in sebha resulted in the presence of only one pubmed indexed article by hassan and one document that was an assignment report on the oral health situation in socialist people's libiyan arab jamahiriya submitted to who geneva by leous . The study by hassan among patients attending the dental clinic in sebha, libyan arab jamahiriya, reported that the people here, have poor oral hygiene and low level of education . Furthermore, it was found that caries (54%) and periodontal disease (41%) were the main causes for the high frequency of tooth extraction . The report by leous was a pathfinder study conducted on 849 children and 269 adults from 14 sites of the following localities: tripoli, sebha, benghazi, zwara, ajelat, and kaddah . The cpitn index was used to assess the periodontal disease, and its results indicate that the periodontal disease was present from a very young age . Very small population in the age group of 20 - 24 years was found to have a healthy periodontium . While drawing a comparison between the population aged 18 years and 20 - 24 years, respective percentage distributions were observed: 3% and 2% had healthy periodontium, 8% and 0% had bleeding, 66% and 69% had calculus, 25% and 27% had shallow pockets, and 0% and 2% had deep pockets . Comparing results of the present study with the results reported by leous indicate the percentages of people with cpi code 0, 1, 3, and 4 to be different, while the prevalence of calculus (code 2) was found to be the highest in both the studies . In the who global oral health data bank for periodontal disease using cpitn index, there was no data available for comparison in the age group 18 - 34 years from emro countries . However, for the ages 18 and 19 years, data from countries belonging to regional office for europe (euro), regional office for western pacific (wpro), regional office for africa (afro), and regional office for south east asia (searo) was available and was compared with the present study participants of the same age [figure 2]. Figure 2 presents a view for all the countries, calculus (code 2) was the highest - recorded cpi score except in south africa where the percentage of healthy periodontium was the highest (51%). Overall, it was observed that the highest prevalence of shallow pockets (34%) was from sebha . Overview of periodontal status from different countries it is widely assumed that symptoms of periodontal diseases escalate with age . Table 3 clearly indicates that the mean number of sextants for lower codes of 0 (healthy) and 1 (bleeding) was highest among 18 years old, but as the age increased, the mean number of sextants for higher codes of 2 (calculus) and 3 (shallow pockets) increased . The presence of shallow pockets, which is an indication of the deterioration of periodontium, was more prevalent in the age group of 25 and above . In the present study, a significant relation was obtained between cpi codes and gender [table 2]. Although, there was an unequal distribution of males and females, the individual percentage calculation showed that more females had healthy periodontium, bleeding, and calculus as compared to males . However, the prevalence of shallow and deep pocket was more prevalent in the males . Studies indicate that predominantly males, because of their smoking habit, have shown more susceptibility to periodontal diseases . Though, the smokers in the present study were all males, significant difference in periodontal status was not observed with smoking . This is a good percentage when compared to other previous studies on libyan and sudanese population, where the percentages were 74.6% and 53%, respectively . Currently, many of the world populations in india, pakistan, several african countries, the arab countries, and most of the muslim world still use miswak . The study reported that the sudanese regular miswak users had better oral health and lower levels of oral pathogens than adult sudanese who used a modern toothbrush regularly . However, in the present study, the frequency of tooth brushing was not significantly associated with the cpi codes . Within the limits of the study, it can be concluded that only 4.7% young adults in sebha have healthy periodontium . The benefits accuring from the control of periodontal diseases and promoting good periodontal health include, improved quality of life, enhanced general well - being and appearance, reduced halitosis, elimination of bleeding gums, reduced potential threat to longevity of teeth, and improved masticatory function . More importantly, ensuring implementation of preventive procedures amongst young adults could possibly lessen the prevalence of periodontal diseases during their adulthood . This study is probably, the first of its kind to report data on the young adults in sebha and this information can be utilized for planning a preventive model that will include dental health education and preventive procedures.
The retinal pigment epithelium (rpe) is a monolayer of pigmented cells situated between the neuroretina and the choroids . The rpe is of neuroectodermal origin and is therefore considered to be part of the retina . The apical membrane of the rpe faces the photoreceptor's outer segments and its basolateral membrane faces bruch's membrane, which separates the rpe from the fenestrated endothelium of the choriocapillaris (figure 1). The rpe constitutes the outer blood - retinal barrier (brb). The inner brb is mainly constituted by endothelial cells . Tight junctions between neighbouring rpe cells and neighbouring endothelial cells are essential in the strict control of fluids and solutes that cross the brb as well as in preventing the entrance of toxic molecules and plasma components into the retina . The main functions of the rpe are the following: (1) transport of nutrients, ions, and water (2) absorption of light and protection against photooxidation, (3) reisomerization of all - trans - retinal into 11-cis - retinal, which is a key element of the visual cycle, (4) phagocytosis of shed photoreceptor membranes, and (5) secretion of various essential factors for the structural integrity of the retina . Apart from these functions, the rpe stabilizes ion composition in the subretinal space, which is crucial for the maintenance of photoreceptor excitability . In addition, the rpe contributes to the immune privileged status of the eye as part of the brb and by the secretion of immunosuppressive factors inside the eye . In recent years it has become clear, mainly from in vitro studies, that rpe cells play an important role in immune responses by the expression of major histocompatibility complex (mhc) molecules, adhesion molecules, fasl and cytokines . With these different complex functions, a failure of any one of these functions can lead to degeneration of the retina, loss of visual function, and blindness . Diabetic retinopathy (dr) remains the leading cause of blindness among working - age individuals in developed countries . Whereas proliferarive diabetic retinopathy (pdr) is the commonest sight - threatening lesion in type 1 diabetes, diabetic macular edema (dme) is the primary cause of poor visual acuity in type 2 diabetes . Because of the high prevalence of type 2 diabetes, dme is the main cause of visual impairment in diabetic patients . In addition, dme is almost invariably present when pdr is detected in type 2 diabetic patients . Neovascularization due to severe hypoxia is the hallmark of pdr whereas vascular leakage due to the breakdown of the blood retinal barrier (brb) is the main event involved in the pathogenesis of dme [7, 8]. Most of the research on the physiopathology of dr has been focused in the impairment of the neuroretina and the breakdown of the inner brb . By contrast, the effects of diabetes on the rpe have received less attention . In the following sections the functions of the rpe mentioned above will be described in more detail, and the deleterious effects of diabetes will be summarized . Although there is growing evidence pointing to rpe as an active secretor epithelium, it seems that this important function has been less recognized . For this reason, this review will focus on this essential propriety of rpe and its impairment in dr . In one direction, the rpe transports electrolytes and water from the subretinal space to the choroid, and in the other direction, the rpe transports glucose and other nutrients from the blood to the photoreceptors . The rpe takes up nutrients such as glucose, retinol, ascorbic acid, and fatty acids from the blood and delivers these nutrients to the photoreceptors . To transport glucose, the rpe contains high amounts of glucose transporters in both the apical and the basolateral membranes . Both glut1 and glut3 glut3 mediates the basic glucose transport while glut1 is responsible for inducible glucose transport in response to different metabolic demands . Another important function of the rpe is the transport of retinol to ensure the supply of retinal to the photoreceptors . The bulk of the retinal is exchanged between the rpe and the photoreceptors during the visual cycle in which all - trans - retinol is taken up from the photoreceptors, isomerized to 11-cis - retinal, and redelivered to photoreceptors . Delivery of fatty acids such as docosahexaenoic acid (dha) to the photoreceptors is a third kind of transport of importance for visual function . Dha is an essential omega-3 fatty acid that cannot be synthesized by neural tissue but is required as structural element by membranes of neurons and photoreceptors . Dha is synthesized from its precursor, linolenic acid, in the liver and transported in the blood bound to plasma lipoprotein where it is taken up in a concentration - dependent manner [1, 14]. Apart from the rpe's functional integrity, dha is the precursor of neuroprotectin d1 (npd1), a docosatriene that protects rpe cells from oxidative stress [15, 16]. Recently it has been demonstrated that high glucose downregulates glut-1 by akt pathway activation mediated by the pkc - oxidative stress signaling pathway in arpe cells (a spontaneously immortalized line of rpe cells). In addition, the transport of retinol may be altered due to a downregulation of the interstitial retinol binding protein (irbp) that occurs in diabetic patients (see below). Finally an impairment of the transport of ascorbic acid also exists in the presence of hyperglycemia, thus limiting the rpe's antioxidant defence [18, 19]. To the best of our knowledge, there is no information regarding the potential effects of diabetes on npd1 or its precursor dha . The rpe transports ions and water from the subretinal space or apical side to the blood or basolateral side . The na - k - atpase, which is located in the apical membrane, provides the energy for transepithelial transport [2023]. There is a large amount of water produced in the retina, mainly as a consequence of the large metabolic turnover in neurons and photoreceptors . Furthermore, intraocular pressure leads to a movement of water from the vitreous body into the retina . This establishes the need for the constant removal of water from the inner retina to the choriocapillaris . Water in the inner retina is transported by mller cells, and water in the subretinal space is eliminated by the rpe [25, 26]. Constant elimination of water from the subretinal space produces an adhesion force between the retina and the rpe that is lost by inhibition of na - k - atpase by ouabain . The transport of water is mainly driven by a transport of cl and k [24, 2830]. Tight junctions establish a barrier between the subretinal space and the choriocapillaris [31, 32]. Paracellular resistance is 10 times higher than transcellular resistance, classifying the rpe as a tight epithelium [33, 34]. For this reason, water cannot pass through the paracellular transport route and water transport occurs mainly by transcellular pathways facilitated by aquaporin-1 [3537]. Recently we have found that high glucose concentrations result in a reduction of permeability in arpe-19 cells that was unrelated to tight junction (occludin, zo-1 and claudin-1) changes . In this regard, in cultured bovine rpe cells it has been demonstrated that hyperglycemia induces a loss of na+/k(+)-atpase function, which responds to aldose reductase inhibitor treatment . Therefore, hyperglycemia could impair the transport of water from subretinal space to the choriochapilaris and, consequently, might contribute to dme development . At present, there is no information regarding the potential effects of diabetes on aquaporin expression in the rpe . The retina is the only neural tissue that has a direct and frequent exposure to light . In addition, the retina is the part of the body that proportionally consumes more oxygen, thus generating a high rate of reactive oxygen species (ros). The rpe is essential in counterbalancing the high oxidative stress that exists in the retina, and it does this by means of three lines of defence . The first line is the absorption and filtering of light . For this purpose, the rpe contains a complex composition of various pigments (i.e., melanin, lipofucsin) that are specialized to different wavelengths and special wavelength - dependent risks [4143]. As enzymatic antioxidants, the rpe contains high amounts of superoxide dismutase [4447] and catalase [45, 48]. As nonenzymatic antioxidants, the rpe accumulates carotenoids, such as lutein and zeaxanthin [42, 43] or ascorbate [42, 49]. Dr is characterized by reduced levels of molecules with antioxidant activity such as glutathione [50, 51], superoxide dismutase (sod) [50, 52], and ascorbic acid [18, 53], thus favouring retinal tissue damage induced by oxidative stress . In vertebrate retina, vision is initiated and maintained by the photolysis and regeneration, respectively, of light sensitive pigments in the disk membranes of the photoreceptor outer segments . This cyclical process depends on an exchange of retinoids between the photoreceptors and the rpe . Light transduction is initiated by the absorption of light by rhodopsin which is composed of a seven transmembrane domain g - coupled receptor protein, opsin, and the chromophore 11-cis - retinal . Photoreceptors lack cis - trans isomerase and, therefore, all - trans - retinal is metabolized into all - trans - retinol and transported to the rpe . In the rpe retinol is reisomerized by means of cis - trans isomerase to 11-cis - retinal and then redelivered to the photoreceptors . The protein rpe65 (retinal pigment epithelium - specific protein 65 kda) is the protein responsible for isomerization of the all - trans - retinaldehyde to its photoactive 11-cis - retinaldehyde and is essential for the visual cycle . In this regard, it has been shown that rpe65 mutations cause severe retinal diseases such as leber congenital amaurosis . There is a great deal of evidence that the transport of retinoids between these cellular compartments is mediated by the interphotoreceptor retinoid - binding protein (irbp), a large glycoprotein synthesized in the photoreceptors and extruded into the interphotoreceptor matrix (ipm) that fills the subretinal space [5658]. Irbp functions to solubilize retinal and retinol, which are otherwise insoluble in water, and mediates the targeting of these compounds and defines transport direction [5962]. This role for irbp is further supported by the observation that irbp is not only present in the ipm but also in endosomes of the rpe . Transport direction is then defined by the rapid turnover of irbp between the ipm and the rpe . Apart from participating in the visual cycle, irbp is important in fatty acid transport and is essential to the maintenance of the photoreceptors [58, 64]. Recently, it has been demonstrated that lower irbp production is an early event in the human diabetic retina and is associated with retinal neurodegeneration [65, 66]. In addition, the content of cellular retinaldehyde binding protein (cralbp), a protein also related to retinoid metabolism, has been found increased in rpe from diabetic subjects with no clinically apparent diabetic retinopathy in comparison with control donors . Another function in the maintenance of photoreceptor excitability is the phagocytosis of shed photoreceptor outer segments [6870]. Photoreceptors are exposed to intense levels of light, thus leading to accumulation of photo - damaged proteins and lipids . Thus, during each day, the concentration of light - induced toxic substances increases inside the photoreceptors . Light transduction by photoreceptors is dependent on the proper functioning and structure of proteins, retinal, and membranes . Therefore, to maintain the excitability of photoreceptors, the photoreceptor outer segments (poss) undergo a constant renewal process [69, 71, 72]. In this renewal process poss the tips of the pos that contain the highest concentration of radicals, photodamaged proteins, and lipids are shed from the photoreceptors . Through coordinated poss tip shedding and the formation of new pos, a constant length of the pos shed pos are digested and essential molecules, such as docosahexaenoic acid and retinal, are redelivered to photoreceptors to rebuild light - sensitive outer segments from the base of the photoreceptors [69, 73]. An impairment of phagocytosis has been described in long term diabetes and, therefore, it is possible that this could also happen to rpe cells . The rpe is known to produce and to secrete a variety of growth factors [7, 75] as well as factors that are essential for the maintenance of the structural integrity of the retina [76, 77] and choriocapillaris . Thus, the rpe produces molecules that support the survival of photoreceptors and ensure a structural basis for the optimal circulation and supply of nutrients . The rpe is able to secrete pigment epithelium - derived factor (pedf) [7, 79, 80], vegf [7, 8185], fibroblast growth factors (fgf-1, fgf-2, and fgf-5) [7, 8691], transforming growth factor- (tgf-) [7, 9294], insulin - like growth factor - i (igf - i) [95, 96], nerve growth factor (ngf), brain - derived growth factor (bdnf), neurotropin-3 (nt-3), ciliary neurotrophic factor (cntf) [97, 98], platelet - derived growth factor (pdgf) [7, 99, 100], lens epithelium - derived growth factor (ledgf), members of the interleukin family [102104], chemokines, tumor necrosis factor (tnf-), colony - stimulating factors (csf), and different types of tissue inhibitor of matrix metalloprotease (timp) [105110]. Among these factors, the rpe secretes pedf [7, 8082], which helps to maintain the retinal as well as the choriocapillaris structure in two ways . Pedf was described as a neuroprotective factor because it was shown to protect neurons against glutamate - induced or hypoxia - induced apoptosis [76, 111, 112]. In addition, pedf was shown to function as an antiangiogenic factor that inhibited endothelial cell proliferation and stabilized the endothelium of the choriocapillaris [7, 81, 82]. These effects on vascularization also play an important role in the embryonic development of the eye [113, 114]. Using pedf - deficient (pedf-/-) mice, it has been confirmed that pedf is an important modulator of early postnatal retinal vascularization and that in its absence retinal vascularization proceeds at a faster rate and is more susceptible to hyperoxia - mediated vessel obliteration . Another vasoactive factor synthesized by the rpe is vegf, which is secreted in low concentrations by the rpe in the healthy eye [7, 83, 86] where it prevents endothelial cell apoptosis and is essential for an intact endothelium of the choriocapillaris . Vegf also acts as a permeability factor stabilizing the fenestrations of the endothelium . In a healthy eye, pedf and vegf pedf is secreted to the apical side where it acts on neurons and photoreceptors whereas most of vegf is secreted to the basal side where it acts on the choroidal endothelium [118, 119]. The pathogenesis of dme remains to be fully understood but vegf and proinflammatory cytokines have been involved in its development . Nevertheless, the balance between angiogenic (i.e., vegf) and antiangiogenic factors (i.e., pedf) will be crucial for the development of dr . In this downregulation of pedf expression by elevated glucose concentration in cultured human rpe cells was also observed . Therefore, strategies in blocking vegf or stimulating pedf have been proposed as new therapeutic approaches for dr . Apart from the factors mentioned above, in recent years new molecules have been found to be synthesized in rpe . Among them, somatostatin, erythropoietin, and apoa1 seem to be of special interest because they could open up new therapeutic strategies for the treatment of dr . Somatostatin (sst) is a peptide that was originally identified as the hypothalamic factor responsible for inhibition of the release of growth hormone (gh) from the anterior pituitary . Subsequent studies have shown that sst has a much broader spectrum of inhibitory actions and that it is much more widely distributed in the body, occurring not only in many regions of the central nervous system but also in many tissues of the digestive tract, including the stomach, intestine, and pancreas . Sst mediates its multiple biologic effects via specific plasma membrane receptors that belong to the family of g - protein - coupled receptors having seven transmembrane domains . So far, five sst receptor subtypes (sstrs) have been identified (sstrs 15). In the setting of this review it must be pointed out that sst is produced by the retina of various species, including humans [125130]. Furthermore, sstrs are also expressed in the retina, with sstr1 and sstr2 being the most widely expressed [127, 131134]. The production of both sst and its receptors simultaneously suggests an autocrine action in the human retina . The amount of sst produced by the retina is significant as can be deduced by the strikingly high levels found in the vitreous fluid . It must be emphasized that the intravitreous level of total proteins is at least 20-fold less than in serum [135, 136]. Thus, the higher intravitreal concentration of a particular protein in relation to its plasma levels strongly suggests an important rate of intraocular production . The main source of sst in humans is rpe . Thus, it has been demonstrated that sst expression and content is higher in rpe than in the neuroretina (figure 2). The main functions of sst for retinal homeostasis are the following (1) sst acts as a neuromodulator through multiple pathways, including intracellular ca signaling, nitric oxide function, and glutamate release from the photoreceptors . In addition, a loss in sst immunoreactivity was found after degeneration of the ganglion cells . It should be noted that retinal ganglion cells (rgcs) are the earliest cells affected and have the highest rate of apoptosis in diabetes [137, 142]. Therefore, the neuroretinal damage that occurs in dr might be the reason for the decreased sst levels detected in the vitreous fluid of these patients . In fact we have recently found that low sst expression and production is an early event in dr and is associated with retinal neurodegeneration (apoptosis and glial activation). Sst may reduce endothelial cell proliferation and neovascularisation by multiple mechanisms, including the inhibition of postreceptor signalling events of peptide growth factors such as igf - i, vegf, epidermal growth factor (egf), and pdgf . Using a mouse model of hypoxia - induced retinopathy, it has been demonstrated that in retinas overexpressing subtype 2 receptor of somatostatin (sst2) neovascularization was lower than in wild type retinas . In addition, also using a mouse model of hypoxia - induced retinopathy it has been observed that retinal neovascularization increased in sst(2)-ko mice . Furthermore, both sstr2- and sstr3- selective analogues directly inhibit retinal endothelial cell growth in vitro [147, 148]. It is worthy of mention that the intravitreal levels of sst lie within the same range as those showing antiangiogenic effect in experimental studies [149151]. Therefore, sst can be considered as a good candidate to be added to the list of the natural inhibitors of angiogenesis . As previously mentioned, various ion / water transport systems are located on the apical side of the rpe, adjacent to the subretinal space, and, indeed, a high expression of sst - r2 has been shown in this apical membrane of the rpe . Nevertheless, the specific mechanisms involved in ion / water transport driven by sst remain to be elucidated . In dr thus, a lower expression of sst has been found in rpe and neuroretina as well as a dramatic decrease of intavitreal sst levels [137, 152154]. As a result, the physiological role of sst in preventing both neovascularisation and fluid accumulation within the retina is reduced, and consequently the development of pdr and dme is favoured [153, 154]. For all these reasons, intravitreal injection of sst analogues or gene therapy has been proposed as a new therapeutic approach in dr . Erythropoietin (epo) was first described as a glycoprotein produced exclusively in fetal liver and adult kidney that acts as a major regulator of erythropoiesis . However, epo expression has also been found in the human brain and in the human fetal retina . In recent years, we have demonstated that not only epo but also its receptor (epo - r) is expressed in the adult human retina (figure 3) [159, 160]. In addition, intravitreal levels of epo are 3.5-fold higher than those found in plasma . The role of epo in the retina remains to be elucidated but it seems that it has a potent neuroprotective effect [161, 162]. In this regard, it has been shown that epo protects cultured neurons from hypoxia and glutamate toxicity [163165], and its systemic administration reduces neuronal injury in animal models of focal ischemic stroke and inflammation [166168]. In addition, it has been demonstrated using an in vitro model of bovine blood - brain barrier (bbb) that epo protects against the vegf - induced permeability of the bbb and restores the tight junction proteins . Since brb is structurally and functionally similar to the bbb, it is possible that epo could act as an antipermeability factor in the retina . In fact, epo was able to improve dme when administered for treatment of anemia in diabetic patients with renal failure . Epo overexpression has been found in both the rpe and neuroretina of diabetic eyes [159, 160]. This is in agreement with the elevated concentrations of epo found in the vitreous fluid of diabetic patients (30-fold higher than plasma and 10-fold higher than in non diabetic subjects). In fact, high intravitreous levels of epo have recently been reported in ischemic retinal diseases such as pdr [159, 172, 173, 175]. In addition, it has been reported that epo has an angiogenic potential equivalent to vegf [173, 176]. Therefore, epo could be an important factor involved in stimulating retinal angiogenesis in pdr . However, intravitreal levels of epo have been found at a similar range in pdr to that in dme (a condition in which hypoxia is not a predominant event). In addition, intravitreal epo levels are not elevated in non diabetic patients with macular edema secondary to retinal vein occlusion . Finally, a higher expression of epo has been detected in the retinas from diabetic donors at early stages of dr in comparison with non diabetic donors, and this overexpression is unrelated to mrna expression of hypoxic inducible factors (hif-1 and hif-1). Therefore, stimulating agents other than hypoxia / ischemia are involved in the upregulation of epo that exists in the diabetic eye . The reason why epo is increased in dr remains to be elucidated but the bulk of the available information points to a protective effect rather than a pathogenic effect, at least in the early stages of dr . There have been several reports on the protective effects of epo in the retina [175, 178185]. In addition, epo is a potent physiologic stimulus for the mobilization of endothelial progenitor cells (epcs) and, therefore, it could play a relevant role in regulating the traffic of circulating epcs towards injured retinal sites . Recruitment of epcs to the pathologic area would be beneficial because their capability of integrating into damaged vasculature can lead to the reendothelization of acellular vessels . It has recently been shown that a reduction of epcs exists in nonproliferarive dr and it has also been demonstrated that epcs from diabetic donors are less effective in repairing damaged vasculature . In this regard, the increase of intraocular synthesis of epo that occurs in early stages of dr (i.e., in nonproliferative dr) can be contemplated as a compensatory mechanism for repairing the damage induced by the diabetic milieu through an increase in epc recruitment . However, in advanced stages of dr (i.e., in the setting of pdr) a dramatic increase of both vegf and mature epcs has been detected . In this setting, epo could potentiate the effects of vegf, thus contributing to neovascularisation and, in consequence, worsening pdr [181, 189]. The potential advantages of epo or epor agonists in the treatment of dr include neuroprotection, vessel stability, and enhanced recruitment of epcs to the pathological area . However, as mentioned above, timing is critical since if epo is given at later hypoxic stages, the severity of dr could even increase . However, in the case of the eye, disease progression is easy to follow without invasive investigation and allows timing of the administration of drugs to be carefully monitored, hopefully resulting in better clinical outcomes . Apolipoprotein a1 (apoa1) has been recently proposed as a key factor for intraretinal reverse transport of lipids, thus preventing lipid accumulation in the retina . In a proteomic analysis of human vitreous fluid we found that apoa1 was highly intraocularly produced in patients with proliferative dr in comparison with non - diabetic subjects . In addition, we have recently shown higher apoa1 (both mrna levels and protein) in the retinas from diabetic donors in comparison with non - diabetic donors (figure 4) [191, 192]. Moreover, apoa1 immunofluorescence was detected in all retinal layers but mrna was more abundant in rpe . This finding suggests that rpe is the main source of apoa1 in the human retina . These results are consistent with those reported by li et al . Which demonstrated the immunolocalization of apoa1 to bruch's membrane (a thin connective tissue between the basal surface of the rpe and the choriocapillaris) in postmortem human eye specimens as well as the presence of apoa1 transcripts in the rpe and neural retina . Several independent lines of research indicate that the rpe contains ldl receptors (ldlrs) and/or scavenger receptors by which lipoproteins (ldl) are internalized and serve as a significant supply of lipids to the retina [194196]. Taken together, the rpe, due to its capacity in internalizing and extruding lipids, can be considered as the most important regulator of lipid transport in the retina . The reason why apoa1 is overexpressed in the diabetic retina needs to be elucidated but one possibility is that the diabetic milieu stimulates apoa1 production by the retina . In this regard, kawai et al . Observed an increased secretion of apoa1 from the main lacrimal gland in patients with dr, but it was not detected in healthy subjects . In recent years new insights have been gained into the transport of lipids within the retina [190, 194], thus allowing us to hypothesize that the mechanisms regulating intraretinal lipid transport rather than serum levels are more important in the pathogenesis of dr [191, 192, 198]. In this regard, abca (atp binding cassette transporter a1) and apoa1 have been found in several layers of monkey retina, thus suggesting the existence of an intraretinal mechanism to export hdl - like particles . Ishida et al . Have demonstrated that hdl stimulates the efflux of radiolabelled lipids, of photoreceptor outer segment origin, from the basal surface of rpe cells in culture . The role of this hdl - based intraretinal lipid transport could be important in preventing lipotoxicity . The fact that the retina is the only neural tissue that has a direct and frequent exposure to light presents a significant problem . This is because many lipids, especially polyunsaturated fatty acids (which are mainly located in the photoreceptor outer segments) and cholesterol esters, are highly susceptible to photo - oxidation and these oxidized lipids become extremely toxic to retinal cells . In dr, this problem could be aggravated by the increase of oxidative stress and lipid peroxidation associated with diabetes . Apart from preventing or arresting lipotoxicity, apoa1 is a potent scavenger of reactive oxygen species [200, 201]; therefore, it could play an important role in protecting the retina from the overall oxidative stress due to diabetes . In this regard, it should be noted that retinopathy has been associated with apoa1 deficiency of genetic origin [202, 203]. Lipoprotein deposition plays an essential role in the pathogenesis of age - related macular degeneration (armd) [204, 205], but little is known about the origin of lipoproteins in the retina of diabetic patients and their potential role in the pathogenesis of dr . The role of apoa1 in extruding lipids out of the retina permits us to hypothesize that apoa1 is increased in diabetic patients as a compensatory mechanism in order to prevent the development of dr . In other words, those diabetic patients with less capacity for apoa1 production by the retina would be more prone to develop lipid deposition (hard exudates) in the retina and, in consequence, to initiate dr . Given that apoa1 has antioxidant properties and prevents lipid deposition in the retina, the design of new treatment strategies addressed to promoting the overexpression of apoa1 in order to reduce the development of dr seems warranted . The rpe lies in the interface between the neural retina and the choriocapillaris where it forms the outer brb . To retard transepithelium diffusion between cells, the cells of the epithelium are bound together by a partially occluding seal, the tight junction . The apical membrane faces the photoreceptors of the neural retina, while the basolateral membrane faces the fenestrated choriochapillaris . As a layer of pigmented cells the rpe absorbs the light energy focused by the lens on the retina . To regulate transport across the monolayer, various pumps, channels, and transporters are distributed specifically to either the apical or the basolateral membrane . The rpe transports ions, water, and metabolic end products from the subretinal space to the blood and, conversely, takes up nutrients such as glucose, retinol, and fatty acids from the blood and delivers these nutrients to the photoreceptors . To maintain photoreceptor excitability retinal is constantly transported from the photoreceptors to the rpe where it is reisomerized to 11-cis - retinal and transported back to the photoreceptors another function that contributes to the maintenance of photoreceptor excitability is the phagocytosis of the shed photoreceptor outer segments . The photoreceptor outer segments are digested, and essential substances such as retinal are recycled and returned to the photoreceptors for rebuilding light - sensitive outer segments from the base of the photoreceptors . In addition, the rpe is able to secrete a variety of growth factors as well as factors that are essential for the maintenance of the structural integrity of the retina and the choriocapillaris . Furthermore, the secretory activity of the rpe plays an important role in establishing the immune privilege of the eye by secreting immunosuppressive factors . Most investigations into the pathogenesis of dr have been concentrated on the neural retina since this is where clinical lesions are manifested . However, rpe is essential for neuroretina survival and, consequently, for visual function . In recent years, various abnormalities in both the structural and secretory functions of rpe have been found in dr . Therefore, future scenarios involving new therapeutic strategies addressed to modulating rpe impairment are warranted.
The national aeronautics and space administration (nasa) has partnered with scientists at the center for food and environmental systems for human exploration of space (cfesh) at tuskegee university to incorporate the sweet potato as a viable dietary item in the space food system . According to nasa, foods in the space system are designed to supply the recommended dietary allowances of the essential nutrients to perform in the space environment . The overall objective of the food processing and product development team (fpd) of cfesh is to convert raw crops such as sweet potatoes into ingredients and nutritious value - added food products for use in space food systems, with applications for consumers on earth . Value - added products developed by fpd include a ready - to - eat sweet potato breakfast cereal [1, 2], a sweet potato bread [3, 4], a sweet potato starch syrup [5, 6], and a sweet potato beverage . Sweet potato (ipomoea batatas (l.) lam .) Is a popular root crop in the united states (usa) and developing countries, which contains roughly 16 to 24% starch . Starches play a vital role in food product development, for example, as raw material, additives, or texture enhancers . Recently, the sweet potato has been increasingly used as an industrial crop for the production of glucose and high fructose syrup . Fpd has used sweet potatoes as a source of starch for syrup production; although a consumer acceptable high glucose syrup has been developed, its storage stability needs to be further improved . A feasible alternative for such improvement is the production of an isomerized fructose syrup due to its noncrystalline nature, ability to prevent microbial growth and extend shelf life . Additionally, fructose is sweeter than sucrose in comparative studies of sweetness, in which the sweetness of sucrose was set at 100, fructose had a sweetness of 173, and glucose had a sweetness of 74 . Gore et al . Reported the commercial manufacture of sweet potato syrup, with the possibilities for use as table syrup, for baking and cooking purposes, and for blending with other syrups to prevent crystallization . At cfesh, silayo et al . Developed an engineering system for converting sweet potato into glucose syrup . Concentration trials utilizing the system produced syrups with volumes of 100 and 70 ml with the respective dextrose equivalence of 281 and 213 mg / ml . Johnson et al . Investigated the feasibility of using native cassava / sweet potato flours and their blends with rice flour and wheat flour, as the raw material for high fructose syrup production . They reported that cassava / sweet potato or their blends with cereal flours had higher fructose yields than native cassava flour and cassava - rice blends . Food products such as syrups contain ingredients that have a major impact on the rheology of the final product . Although researchers have reported the development of sweet potato syrups, very few, if any, have examined its rheological properties . Knowledge of the rheological characteristics of the sweet potato starch syrup (spss) is valuable in predicting pourability and the ease with which it may be handled, processed, or used . Rheological factors play an important role in consumers' selection and continued use of a syrup . Products such as pancake syrup which appear too thick or too runny have little appeal to consumers . Data regarding the effect of storage on the rheological and thermal behaviors of an isomerized spss are limited . This is one of few, if any, studies to report on the rheological and thermal properties of a spss during storage . The study was part of the larger cfesh and fpd project to convert raw crops such as sweet potatoes into ingredients and nutritious value - added food products for use in food systems in space and on earth . The overall objective of this study was to improve the quality and storage stability of an isomerized spss . Specifically, the study isomerized a high glucose spss and determined its sugar profile, mineral content, and rheological and thermal properties . Sweet potatoes (variety: mississippi red) purchased from the local grocery store were utilized to develop the spss . Sweet potatoes were washed free of dirt, weighed, hand - peeled, and shredded . Our laboratory has utilized other varieties of sweet potato in syrup production with good outcomes . For example, earlier studies by miller (2003) and ibrahim (2004) utilized hillbilly and whatley / loretan (hydroponic and field - grown) sweet potatoes in syrup production . Briefly, the shredded roots were weighed and homogenized at 1: 1 water / sweet potato ratio . The resulting starch was ground in a laboratory blender at high speed for 30 seconds . After grinding, the starch was milled, vacuum - sealed, and stored at room temperature (22 2c) until further use . Food - grade enzymes, spezyme fred (thermostable -amylase), optidex l-400 (glucoamylase), gensweet sgi (glucose isomerase), and optimax l-1000 (pullulanase), were supplied by courtesy of genencor danisco division (rochester, new york, usa). The enzymes had the following activities: spezyme fred an activity of 17,400 lu / g (lu is liquefon units); optidex l-400 a minimum activity of 350 gau / g (glucoamylase unit); and optimax l-1000 an activity of 10001260 aspu / g (acid stable pullulanase units). Sweet potato starch (30 g) was mixed with 400 ml distilled water, and the solution was heated to 100c to promote gelatinization of the starch . The ph of the starch was adjusted to 4.5 with naoh or hcl as needed . Upon gelatinization, -amylase (4.5 ml) the solution was then incubated at 90c for 2 hours and allowed to cool to 62c . The cooled solution was treated with 300 l glucoamylase and 300 l pullulanase, incubated at 62c for 12 h to induce glucose formation from the liquefied starch . The ph of the solution was adjusted to 7.5 and 200 l glucose isomerase was added . The solution was then kept in a thermostatic water bath at 60c for 5 h, after which it was concentrated to 6373.9brix . The syrup was measured, bottled, labeled, and stored at room temperature (22 2c) for further evaluation . The samples evaluated were the isomerized spss and two commercial syrups (pancake and ginger). Karo brand pancake (corn) syrup was purchased from a local grocery store, and ginger syrup was provided by courtesy of buderim ginger (mahwah, new jersey, usa). The sugar profile of the syrups was tested at medallion labs (minneapolis, minnesota, usa). The sugars were extracted, diluted, and quantitated using high performance liquid chromatography (hplc). Calcium, magnesium, phosphorus, iron, and potassium contents of the syrups were tested at medallion labs (minneapolis, minnesota, usa). Inductively coupled plasma (argon plasma) was done on the solutions and quantitations were provided by a standard curve . Aliquots (10 ml) of the spss were stored in plastic bottles, sealed, and stored at room temperature (22 2c) for 70 days . Samples were randomly withdrawn for testing on days 0, 15, 30, 49, and 70, and rheological measures were conducted . However, data from day 15 were excluded from the analysis because of technological difficulties with the equipment that day, and data were considered unreliable . Commercially available pancake and ginger syrups were also tested . Steady viscosity, shear rate and shear stress testing were conducted using the ta instruments ar-2000 rheometer (new castle, delaware usa) and modifications of the procedures were described by ngadi and yu . Tests of viscosity versus shear rate and viscosity versus shear stress as a function of time were conducted at a fixed temperature (25c), and the shear rate and shear stress were varied . The rheometer was calibrated to 1000 m minimum gap space; the syrup samples were spread across the bottom plate, and the gap height was set at 4000 m . Spss, pancake, and ginger syrups were tested using the methods described by budke et al . . A hard clear plastic surface was marked with concentric circles spaced 0.5 cm apart from a 2.5 to 7.5 cm radius . The syrup samples were held in a hollow cylinder positioned at the center of the circles for a setting time of 10 minutes . The cylinder was then lifted to allow the sample to spread for 60 seconds . The mean measurement, averaged across bisecting lines at the four quadrants, represented the degree of thickness of the syrup . Lst was conducted on spss, pancake, and ginger syrup on the same days as the instrumental tests were conducted . Thermal scanning of the syrup samples was carried out using the mettler dsc 822e (columbus, ohio, usa), equipped with a refrigerated cooling system that efficiently controlled and monitored temperature up to 250c . Approximately 25 mg of the syrup samples was weighed into aluminum pans, which were hermitically sealed and placed in the dsc . One - way analysis of variance (anova) at a 0.05 level of probability was used as the criterion of significance for the sugar profile and mineral content tests . Repeated measures anovas were used to (1) compare the viscosity measures (instrumental and lst) of the spss, pancake, and ginger syrups throughout storage and (2) compare the thermal measurements of spss, pancake, and ginger syrups to determine whether there were differences among the syrup samples during storage, and fisher's lsd (p <0.05) was used to determine where those differences lie . Pearson correlations were used to assess the nature of the relationship between the instrumental and lst measures . The starch content of the sweet potato was roughly 81% and the ratio 147% amylose: 8693% amylopectin ratio . The glucose syrup produced from the spss was concentrated to 6373.9brix and was then isomerized at 60c for 5 h using glucose isomerase . Prior to isomerization, the spss had a glucose content of 51% and <1.0% fructose . The intention was to improve the fructose content by extension of the sweetness and storage stability of the spss through isomerization . After isomerization, although the fructose yield was slightly improved, the major sugar found in the spss was glucose (47.2 1.8%). Perhaps, in this study, the incubation time was too short for isomerization of glucose to fructose . Our study used 60c and 5 h incubation time, whereas johnson et al . (2009) used 60c and 48 h incubation time for isomerization to a high fructose sweet potato syrup . It should be noted that the fructose yield in the current study was higher (7.6 or 7.6/100 g versus 5.7 or 5.7/100 g) than johnson et al . 's, who used direct conversion of sweet potato roots and not the isolated starch . Furthermore, older studies reported that the enzyme requires magnesium and cobalt as activators; neither of these were used in the current study . The spss had higher amounts of calcium than the pancake and ginger syrups (table 1). It has been reported by crabb and shetty that it is preferable to remove calcium ions prior to isomerization because they inhibit the isomerization of glucose into fructose . The possible inhibition of glucose isomerase by calcium ions may be due to the competition with the required magnesium ions for the enzyme's active site . It has been reported by kasumi et al . That the enzyme glucose isomerase depends on either magnesium or manganese ion for its activity to take place . It is speculated that the ca content of the spss negatively affected the isomerization process of glucose to fructose in the syrup . The mean calcium content (32.9 0.3 mg/100 g) of the spss was significantly (p <0.05) higher than that of the pancake and ginger syrups . The calcium content was roughly eight and 16 times more in the spss than that in the ginger and pancake syrups, respectively . The high calcium content of the spss could have been due to the typically high calcium content of sweet potatoes, although calcium content of different varieties of sweet potatoes could vary greatly . The average amount of magnesium in the spss was 23.4 0.6 mg/100 g (table 1). Similar to the calcium, the magnesium content of the spss was significantly (p <0.05) higher than the two other syrups, which had negligible amounts of magnesium . The pattern was similar for phosphorus; the spss had a significantly (p <0.05) higher phosphorus content (49.7 0.1 mg/100 g) than the pancake and ginger syrup (table 1). The iron contents of spss and ginger syrup were similar with an average of 0.5 0.0 and 0.6 0.0 mg/100 g, respectively, but the pancake syrup had significantly (p <0.05) less iron (table 1). Likewise, the mean potassium content of the spss was 268 8.5 mg/100 g (table 1), which was significantly (p <0.05) higher and several times more than that of the pancake and ginger syrups . Overall, the results of this study demonstrated that the spss had higher mineral (calcium, magnesium, phosphorus, iron, and potassium) content when compared to the ginger and pancake syrups . In general, as shear rate increased the apparent viscosity of the spss, pancake, and ginger syrups decreased, which resulted in the syrups exhibiting shear - thinning behavior as described by abu - jdayil et al . . These shear - thinning effects indicated that the spss and other syrups behaved as non - newtonian fluids . In shear - thinning fluids, the shear - thinning behavior observed in the three syrup samples could be attributed to stress - induced breakdown of the network structure ., the phenomenon of shear thickening was not observed in the syrup samples used in the current study . The flow curves of the spss, pancake and ginger syrups are shown in figures 2(a)2(d). It can be seen in figure 2(a) that, after a sharp reduction, the viscosity was stabilized at high shear rates . This result suggests that the onset of shear thinning occurs at a lower shear rate . It has been speculated that this could be due to the onset of entanglements caused by the presence of the highly branched, high molecular weight amylopectin . Sweet potatoes contain high amounts of the branched - chain polymer amylopectin, with branch points occurring through -1 - 6 bonds . Generally, amylopectins occur in most cereals and plants, contributing to about 80 to 85% of the total starch content . According to kelco, high viscosity is desirable only under low shear rate conditions and should decrease as flow rate increases . Spss, pancake, and ginger syrup had their highest viscosities under the lowest shear rates, and viscosity of the syrups decreased as flow increased . Viscosity readings of the three syrups were observed at 65 seconds for each trial . On day 0 however, on days 30, 49, and 70, viscosities of the syrups were significantly (p <0.05) different . On day 30, all the three syrups were significantly (p <0.05) different from one another . The pancake and ginger syrups had similar viscosities on days 49 and 70 but were significantly (p <0.05) different from the spss . The mean viscosity of the spss, pancake, and ginger syrup increased between days 0 and 30 and decreased between days 49 and 70 . Strain is imposed on syrup when it is poured into bottles for storage and also when the syrup is poured unto waffles or pancakes by consumers; therefore data from this study confirmed that the viscosity of spss, pancake, and ginger changed over time as strain was applied . Typical shear rates versus shear stress responses of the spss, pancake, and ginger syrups at the constant temperature of 22c are shown in figures 3(a)3(d). As expected, shear stress increased with increasing rate in a fairly linear fashion . The mean apparent viscosity ranged from 0.03 to 0.50, 0.01 to 14.2, and 0.07 to 2.2 pas for spss, pancake, and ginger syrups, respectively, during storage . In general, scarce information is available in the published literature regarding the rheology of sweet potato syrup . However, ngadi and yu reported apparent viscosities ranging from 0.035 to 0.651 pas for different grades and temperatures of maple syrups . Johnson also reported the viscosity of maple syrup at 25c as 0.1635 pas . The viscosity for spss at 25c (0.03 to 0.5 pas) in the current study was consistent with the range obtained for maple syrup . On days 0, 30, and 70, ginger syrup had the highest viscosity while, on day 49, compared to the results for days 0, 30, and 70, the pancake syrup displayed unusual elevations in viscosity on day 49, even though extreme care was taken while conducting the experiment and measures were done in duplicate with the intent of minimizing errors . It is still possible that, despite the care taken, some type of experimental error influenced the result for the pancake syrup on day 49 . Overall, the spss and pancake syrup were the thinner syrups with similar viscosities on days 0 and 70 . Viscosity readings of the spss using lst were similar to the instrumental rheological data . As with the instrumental data, on day 0, the viscosities of the three syrups were similar . Pancake and ginger syrup had a significantly (p <0.05) higher viscosity than spss on day 30 . On days 49 and 70, pancake and ginger syrups were similar to each other . At the beginning of the storage study there was a weak positive relationship (r = 0.4) between the line spread and instrumental measures . However, on days 30, 49, and 70, the relationship between the line spread and instrumental measures was strong (r = 0.8, r = 0.7, and r = 0.9, resp . ). It is unclear why the results from day 0 of line spread and instrumental measures were poorly correlated . We can only speculate that the viscosities of the syrups were not yet stabilized on day 0, which resulted in its poor correlation . The water loss curves obtained during dsc scanning of the spss, pancake, and ginger syrup samples from day 0 are presented in figure 4 . Each syrup sample showed an endothermic curve which indicates that the syrups absorbed energy during heating . Water loss temperatures and melting endotherms of syrups are fairly broad because sugars do not have sharp water loss temperatures and their water loss proceeds over a temperature range . Water loss temperatures were recorded as the transition peak, which were found to vary among the syrup samples from day to day . Water loss temperature of the spss increased from days 0 to 30 and then decreased from days 30 to 70 . Pancake and ginger syrups' peak water loss temperature decreased from days 0 to 49 and then increased from days 49 to 70 (table 2). Sugar concentrations of the syrups could reflect the water loss temperature . As stated previously, the increase in water loss temperature from days 49 to 70 for pancake and ginger syrups could be related to the fact that they had higher fructose contents than the spss . Ginger syrup had the highest water loss temperature of all syrups and also the highest percentage of fructose . The water loss temperatures of spss, pancake, and ginger syrups were similar during the storage study . This result indicates that the water loss temperature of the spss was similar to the commercial pancake and ginger syrups . This study lasted for only 70 days and 60c was the lowest recorded water loss temperature for spss . It would require a longer time study to dictate whether or not the water loss temperature goes below 60c . The present study utilized the batch process for syrup production because of available laboratory equipment . In the production of a corn - based high fructose syrup (hfs), a continuous process is usually used, which is more time and energy efficient than the batch process . However, if the continuous process is employed for the production of spss, the authors believe that it will be much more economically feasible than the corn - based hfs . In terms of sensory analysis, ibrahim (2004) determined the acceptability of the sweet potato syrup in 112 sixth grade students . The isomerized spss has been tested in college - age students; the dataset is being analyzed . Thermostable -amylase, glucoamylase, glucose isomerase, and pullulanase were used to hydrolyze sweet potato starch into syrup with comparable, and in some instances superior nutritional, rheological and thermal properties to commercially available ginger and pancake syrups using the enzymes . Our study has added important information to the knowledge base regarding the rheological and thermal properties of the spss . Additionally, the spss had significantly higher mineral content than the ginger and pancake syrups, making it a much more nutritious option . Further and more detailed studies should be designed to further enhance the fructose content of the syrup and observe its stability beyond 70 days . Overall, the spss has the potential to be used in food systems in space and on earth.
Simultaneous occurrence of hashimoto's thyroiditis (ht), and graves disease (gd) is rare . A 60-year - old lady presented with tremulousness of hands, palpitation, and excessive sweating . Her weight was 46 kg, bmi 17, afebrile, regular pulse rate of 110/min with fine tremor in hands . Thyroid gland was symmetrically enlarged, firm, without any bruit, but mildly tender with lobular surface . Initial thyroid function tests (tft) revealed: t3: 3.80 ng / ml (0.80 - 2.10), t4: 12.40 ug / dl (5.10 - 12), thyroid stimulating hormone (tsh): 0.20 u / l (0.70 - 5). Her anti thyroperoxidase (tpo) antibody: 374 iu / ml (normal [nl .] <35) and tsh receptor antibody: 15 u / l (nl . <1) were both strongly positive . 99mtc pertechnetate scan showed an enlarged gland with increased uptake of radiocontrast: 17% (nl . Thyroid fine needle aspiration cytology (fnac) showed sheets of hurthle cells with abdunce of lymphocytes indicating ht . Repeat tft, 3 months later showed: t3: 4.20 ng / ml, t4: 14.40 ug / dl, tsh: 0.001 u / l, with increased uptake on repeat scan . Our case of an elderly lady with no eye signs, lobular, firm tender goiter with patchy uptake in both lower poles on tc99 m scan were odd points in diagnosing isolated gd . Hashimoto's thyroiditis (ht) and graves disease (gd) are the two main types of autoimmune thyroid disease . Ht rarely occurs following gd . But combined occurrence of gd and ht are rare . A 60-year - old lady, presented with tremulousness of hands for 1 month and palpitation with excessive sweating for 3 months . She had a history of weight loss for 6 months, and gradually enlarging neck swelling for 3 years . Her weight was 46 kg, bmi 17, afebrile, pulse rate: 110/min / regular, blood pressure: 140/56 mm of hg . Her thyroid gland was symmetrically enlarged, firm in consistency, nodular / lobular surface and mildly tender without any bruit . Initial thyroid function tests (tft) revealed high t3: 3.80 ng / ml (0.80 - 2.10), t4: 12.40 ug / dl (5.10 - 12) with a low tsh: 0.20 mu / l (0.70 - 5). Her tsh receptor antibody (tsab): 25 u / l (normal [nl .] <1), anti tpo antibody: 374 iu / ml (nl . <35) and thyroglobulin antibody: 268 iu / ml (nl . <65) were all strongly positive . Ultrasonogram of thyroid revealed an enlarged gland with hypoechoic parenchyma with fibrous septa.tc pertechnetate scan (tc99 m scan) revealed enlarged thyroid gland with diffusely increased uptake of radiocontrast: 17% (nl . Thyroid fnac showed sheets of hurthle cells with abdunce of lymphocytes and sheets of regular follicular cells, indicating ht . Repeat tft, 3 months later showed: t3: 4.20 ng / ml, t4: 14.40 ug / dl, tsh: 0.001 u . Repeat tc pertechnetate scan was done, which again revealed enlarged thyroid gland with diffusely increased uptake of radiocontrast: 21% (nl . Our patient presented with clinical symptoms and signs of thyrotoxicosis along with slightly raised t4, t3 and low tsh with positive tsab, and diffusely increased uptake on tc99 m scan, all in favor of a diagnosis of gd . However, there were some odd features such as elderly age, absence of eye signs, nodular, firm mildly tender goiter, hypoechoic parenchyma in ultrasonography (usg), and patchy uptake in both lower poles on tc99 m scan . Fnac of the thyroid gland was suggestive of ut fnac of thyroid gland suggested ht . So we adopted a wait and watch policy, and put the patient on beta blockers . Repeat tft, 3 months later showed persistent thyrotoxicosis and repeat tc99 m scans revealed an enlarged thyroid gland with diffusely increased uptake of radiocontrast . Diffusely increased uptake on tc99 m scan rules out hashitoxicosis, which mostly resolves within 3-months time - frame . A diagnosis of combined occurrence of hyperthyroid gd and hd was established and anti - thyroid medication started . Reported four cases of ht in previously diagnosed patients with gd hyperthyroidism . In three cases, ht occured 7 - 25 years after gd treatment; in one, it developed in a few months of gd treatment . The diagnosis of ht was based on clinical manifestation, positive tpo and thyroglobulin antibody, and fnac . All were middle - aged women, who had high titers of anti - thyroid antibodies and thyrotoxicosis at the onset of painful ht . Reported seven patients with hypothyroidism due to hashimoto's disease, who developed hyperthyroid gd . Some had transient, some persisistent hyperthyroidism due to gd following hypothyroidism due to ht . Ht and gd represent the main two types of autoimmune thyroid disease . Gd is caused due to hyperplasia and hyperfunction of the thyroid gland by stimulating tsab . On the contrary, ht is thought to be due to a tsh stimulation - blocking antibody (tsbab) which blocks the action of tsh hormone and subsequently brings damage and atrophy to the thyroid gland . Alterations in the thyroid state related to the balance between the activities of tsab and tsbab . Moreover, approximately 15 - 20% of patients with gd have been reported to have spontaneous hypothyroidism resulting from the chronic ht . Patients with gd who have achieved remission on anti - thyroid drugs may also experience ht or hashitoxicosis or intermittent transient hyperthyrodism . They may have both ht and gd since antibodies associated with both diseases may be present . To distinguish from relapsed gd a wait and see approach is beneficial . A definitive diagnosis can be made by fnac biopsy, that is, fine - needle aspiration . Hashimoto's disease, which occurs following gd may be due to extended immune response to endogenous thyroid antigens, i.e. Thyroid peroxidase and thyroglobulin, which may enhance lymphocyte infiltration and finally cause ht . In conclusion, ht rarely occurs following gd . In our case of an elderly lady, absence of eye signs, lobular - firm tender goiter with patchy uptake in both lower poles on tc99 m scan were odd points in diagnosing isolated gd.
Plasma cell gingivitis (pcg) is a rare condition characterized by diffuse and massive infiltration of plasma cells into the sub - epithelial gingival tissue . Clinically, it appears as diffuse reddening and edematous swelling of the gingiva with a sharp demarcation along the mucogingival border . Pcg is known by a variety of other names such as atypical gingivitis, plasma cell gingivostomatitis and allergic gingivostomatitis . The etiology of pcg is not clear, but due to the obvious presence of plasma cells many authors are of the opinion that it is an immunological reaction to allergens . Flavoring agents such as cinnamonaldehyde and cinnamon in toothpaste and chewing gum, mint pastels, khat, strong spices and some herbs such as chili, pepper and cardamom may be important factors . Aggressive periodontitis (agp) represents the most heterogenous and severe form of periodontitis due to its peculiar clinical presentation: occurring around puberty and amount of microbial deposits inconsistent with the severity of periodontal destruction . This report outlines a case of pcg associated with generalized aggressive periodontitis (gagp), which was brought on by the use of herbal toothpaste containing acacia extract from the tree acacia arabica . A 17-year - old girl was referred to the department of periodontics with the chief complaint of gingival overgrowth and mobility in her teeth for the last 2 years . She reported that the problem has begun in lower anterior and progressed involving both the arches . Patient appeared fit and indicated no current or previous systemic disease during her medical history interview . Intraorally, severe diffuse gingival enlargement of both the arcades (more pronounced on the right side) was observed covering almost all the surfaces of teeth and projecting into the vestibule [figure 1a c]. The erythema was disproportionate to the amount of plaque and calculus remaining on the dentition panoramic radiograph demonstrated generalized alveolar bone loss [figure 2]. Probing depth ranged from 10 mm to 12 mm with an attachment loss of 7 - 9 mm and was more in the right molar region . Grade 2 mobility was present around the mandibular and maxillary first and second molars and grade 1 around the mandibular right premolars . (a) facial; (b) maxillary occlusal and (c) mandibular occlusal view of enlargement of the gingiva at the initial visit panoramic radiograph at the initial visit laboratory tests revealed no evidence of any systemic disease such as leukemia, scurvy and hormonal disorders . Internal bevel gingivectomy was performed in the maxillary arch and left mandibular region[figure 3]. Adjunctive amoxicillin (500 mg, 3 1) plus metronidazole (400 mg, 3 1) for 7 days was prescribed to treat agp . Patient could not continue her treatment for personal reasons and when she reported after 1 year, the recurrence of gingival enlargement was noticed [figure 4a c]. Periodic panoramic radiographic examination revealed progressive alveolar bone loss, which was more pronounced on the right lower molars [figure 5]. Sub - epithelial tissue revealed a large number of normal plasma cells, few eosinophils and inflammatory granulation tissue [figure 6a and b]. A diagnosis consistent with pcg was made . To clarify whether this enlargement was due to a hypersensitivity reaction the patient was questioned about the habitual use of chewing gum, mouthwash and toothpaste . The only relevant history given by the patient was the use of herbal toothpaste (babool, dabur oral care products, india) for the last 3 - 4 years . The patient was advised to discontinue the use of herbal toothpaste, which dramatically reduced the severity of enlargement within 2 weeks [figure 7a c]. Moreover, the patient herself switched on to other herbal toothpaste (meswak, dabur oral care products, india) after discontinuing babool toothpaste . Acacia arabia was responsible for the gingival enlargement in the present case as all other constituents of both the herbal toothpastes are similar . Facial view of the gingiva after periodontal treatment (gingivectomy in the maxillary arch and left mandibular region) (a) facial; (b) maxillary occlusal and (c) mandibular occlusal view showing recurrence of gingival enlargement 12 months post - surgery panoramic radiograph 1 year after the initial visit (a) biopsy comprised of plasma cell gingivitis with hyperplastic stratified squamous epithelium with dense plasma cells infiltrate in sub - epithelium (h and e, 200); (b) high power magnification revealing abundant normal plasma cells (h and e, 400) (a) facial, (b) maxillary occlusal and (c) mandibular occlusal view of regression of enlargement after discontinuation of herbal toothpaste blood test (enzyme - linked immunosorbent assay) for allergy of toothpaste components disclosed very high level of allergen specific antibody and symptoms relation for acacia (105 kua / l; normal value <0.35 although pocket depth reduced significantly from 10 - 14 mm to 6 - 8 mm, but clinical attachment level was not reduced significantly . Surgical procedures were carried out on right molar region to treat agp and residual enlargement . Patient was recalled at 1, 3, 6 months and after that she was recalled at 6 months interval . No recurrence of gingival enlargement was noticed in both arches and probing depth ranged from 3 mm to 5 mm [figure 8]. No recurrence of gingival enlargement was seen panoramic radiograph after 1 year follow - up showing no further progression of bone loss the initial presentation of severe gingival enlargement associated with gagp necessitated the formulation of an extensive differential diagnosis . Clinically, the enlarged gingiva in idiopathic gingival fibromatosis is firm and leathery in consistency and the histologic features are densely arranged collagen bundles and numerous fibroblasts; both of which differ from the features in this patient . Plaque induced gingivitis would normally involve the marginal gingiva alone and not the entire width of attached gingiva . In the present case, there was an inflammation of marginal and attached gingiva, which was not responding to local therapy and hence inconsistent with a plaque related etiology . Gingival hyperplasia is well - known consequence of administration of certain drugs, such as phenytoin, cyclosporine and nifedipine . Gingival enlargement due to scurvy was ruled out as the patient did not have any signs of scurvy, such as petechiae, ecchymoses, or spontaneous bruising of the extremities . The presence of a large number of plasma cells can occasionally lead to a difficulty in distinguishing pcg from more exotic and rare plasma cell lesions affecting the gingiva such as extramedullary plasmacytoma, plasmacytosis of the gingiva and plasma - cell granuloma . Pcg is purely benign and the detection and elimination of exposure to the etiologic antigenic agent will bring about remission of the condition . However, sheets of atypical plasma cells may represent multiple myeloma, waldenstrm's macroglobulinemia and solitary plasmacytoma . Pcg is frequently associated with cheilitis and glossitis and sometimes with psoriasis, but the patient did not exhibit any of these symptoms . Marker and krogdahl sub - divide pcg into three types: (1) caused by an allergen, (2) neoplastic and (3) unknown cause . Reached the conclusion that pcg is the result of an allergic reaction to bacterial plaque, even though this had been eliminated by means of conventional periodontal treatment . Reported marked inflammatory gingival enlargement with a predominance of plasma cells associated with rapidly progressive periodontitis, but were not able to identify the cause of the gingival enlargement . The present case belongs to type 1 as the changes had developed after prolonged use of herbal toothpaste containing acacia for last 2 years . Amelioration of the gingival enlargement was obtained to a large extent when the use of the toothpaste was discontinued . Acacia tree is a thorny, scraggly evergreen tree, native to africa, southern california and the indian subcontinent . Whole gum mixtures of acacia have been shown to inhibit the growth of periodontopathic bacteria, including porphyromonas gingivalis and prevotella intermedia in vitro . A significant number of this genus of plants bears pollen and has been reported to cause pollinosis or strong allergic reactions . Extract used in herbal toothpaste as an allergen, which caused severe gingival enlargement was the important finding in this case . Clinically, gagp is characterized by generalized interproximal attachment loss affecting at least three permanent teeth other than first molars and incisors . Radiograph often shows progressive bone loss in spite of the apparent lack of local factors . All these features were identifiable in the patient, so the diagnosis of gagp in association with pcg was confirmed . In many types of gingival enlargement, the rapid loss of alveolar bone or attachment level is not commonly observed . In the present case, pcg was associated with gagp, but it was difficult to retrace the clinical presentation and identify which of the two occurred first . Gingival enlargement as an allergic response to acacia containing herbal toothpaste may be an aggravating factor resulting in exaggerated progressive bone loss . Deepened gingival pockets due to unusual gingival enlargement may have allowed subgingival periodontopathic bacteria to colonize and overgrow and the alveolar bone loss might then advance very rapidly . This case underscores the necessity of a comprehensive history, examination and appropriate diagnostic tests in order to arrive at a definitive diagnosis and treatment plan for gingival conditions, which are refractory to conventional therapy . The importance of this lesion is that it may cause severe gingival inflammation, discomfort and bleeding and may mimic more serious conditions . Hence, the etiologic agent must be identified and the substance should be eliminated from its use . This report outlines a case of pcg which was brought on by the use of herbal toothpaste containing acacia extract from the tree acacia arabica associated with agp.
G - quadruplexes (g4), secondary dna structures consisting of g - quartet planers in g - rich regions, play significant biological roles for example, control of transcription and telomeric lengths [119]. One typical g4 forming dna sequence is a telomere, which exists at the ends of chromosomes consisting of (ttaggg)n repeating single - stranded sequences [112]. Telomeres protect chromosomes from end to end fusion events, which result in replication of the chromosome (the hayflick limit). The telomere repeats are elongated by the reverse transcriptase telomerase, which is overexpressed in most tumor cells . Since the activity of this enzyme is inhibited by the g4 structure of telomeres owing to steric hindrance, small molecules that selectively bind and stabilize the telomeric g4 should be potential anticancer agents . As a result, a number of g4 ligands, inspired by artificial dna intercalators as well as natural products, have been developed during the past decade . Telomestatin (tms) is a natural product isolated from streptomyces anulatus 3533-sv4, which displays one of the most potent telomeric g4 binding activity (figure 1) [2328]. Interaction analysis has shown that two molecules of tms induce conversion of telomeric g4 into an antiparallel type by way of an end stacking mode [2528]. We have recently developed macrocyclic hexaoxazole compounds 6otd, containing a variety of side chain functional groups, that serve as a novel tms derivative [2932]. In addition, by considering the proposed binding mode of tms with telomeric g4, we have carried out further structural development of dimeric 6otd derivatives (figure 1). The results of molecular dynamics calculations guided the selection of 6otd dimer 1 that contains an appropriate length of a linker between the monomeric units of 6otd . Studies showed that dimer 1 binds to telomeric g4 more tightly than do other 6otd dimers with linkers of shorter or longer lengths . One possible structural development strategy to enhance the stabilizing ability of 1 against the g4 would be to install cationic functional groups on the side chain . Below, we describe synthesis of new 6otd dimers 2 and 3 that derivatize 1 but possess cationic amine and guanidine functional groups on their side chains . In addition, the ability of these substances to stabilize telomeric g4 along with their interaction mode was investigated . Flash chromatography was performed on silica gel 60 (spherical, particle size 0.040 ~ 0.100 m; kanto). Optical rotations were measured on a jasco p-2200 polarimeter, using the sodium d line.h andc nmr spectra were recorded on jeol jnm - ecx 300, 400, and 500 . The spectra are referenced internally according to residual solvent signals of cdcl3 (h nmr; = 7.26 ppm, c nmr = 77.0; ppm) and dmso d 6 (h nmr; = 2.50 ppm, c nmr; = 39.5 ppm). Data for h nmr are recorded as follows: chemical shift (, ppm), multiplicity (s, singlet; d, doublet; m, multiplet; br, broad), integration, and coupling constant (hz). Data for c nmr are reported in terms of chemical shift (, ppm). Mass spectra were recorded on a jeol jms - t100x spectrometer with esi - mass mode using methanol as solvent . All oligonucleotides purified were obtained from sigma genosys and dissolved in double - distilled water to be used without further purification . Fluorescence resonance energy transfer (fret) melting assay was made with an excitation wavelength of 470505 nm and a detection wavelength of 523543 nm using the dna engine opticon 2 real - time cycler pcr detection system (biorad). Cd spectra were recorded on a jasco-810 spectropolarimeter (jasco, easton, md) using a quartz cell of 1 mm optical path length and an instrument scanning speed of 500 nm / min with a response time of 1 s, and over a wavelength range of 220320 nm . . Synthesis of 5to a solution of trioxazole 4 (2.1 g, 3.6 mmol) in meoh - thf (1: 1, 60 ml) was added pd(oh)2/c (420 mg), and the reaction mixture was stirred at room temperature under hydrogen gas (balloon). After 3 h, the catalyst was removed by filtration through a pad of celite, and the filtrates were concentrated in vacuo to give amine 5, which was used without further purification . To a solution of trioxazole 4 (2.1 g, 3.6 mmol) in meoh - thf (1: 1, 60 ml) was added pd(oh)2/c (420 mg), and the reaction mixture was stirred at room temperature under hydrogen gas (balloon). After 3 h, the catalyst was removed by filtration through a pad of celite, and the filtrates were concentrated in vacuo to give amine 5, which was used without further purification . Synthesis of 7to a solution of trioxazole 6 (2.1 g, 3.6 mmol) in thf - h2o (3: 1, 80 ml) was added lioh (230 mg, 5.4 mmol) at 0c . After stirring at room temperature for 1 h, to the resulting mixture was added 1 n hcl, to give carboxylic acid 7, which was used without further purification . To a solution of trioxazole 6 (2.1 g, 3.6 mmol) in thf - h2o (3: 1, 80 ml) was added lioh (230 mg, 5.4 mmol) at 0c . After stirring at room temperature for 1 h, to the resulting mixture was added 1 n hcl, to give carboxylic acid 7, which was used without further purification . Synthesis of 8to a solution of amine 5 (abovementioned) in thf - h2o (1: 1) was added the carboxylic acid 7 (abovementioned), n - methylmorpholine (1.2 ml, 11 mmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (dmt - mm) (3.0 g, 11 mmol), and the mixture was stirred at room temperature for 15 h. to the reaction mixture was added h2o and precipitate was formed . This precipitate was collected with filtration using filter paper, to give 8 as a white solid (3.3 g, 3.2 mmol 89% 2 steps, mp = 200203c). Spectral data for 8: []d = 2.7 (c 1.1, chcl3meoh (1: 1)); h nmr (300 mhz, cdcl3) 8.43 (s, 1h), 8.358.27 (m, 5h), 7.53 (d, j = 8.9 hz, 1h), 7.34 (m, 5h), 5.985.81 (m, 1h), 5.575.44 (m, 2h), 5.30 (d, j = 18 hz, 1h), 5.21 (d, j = 11 hz, 1h), 5.154.97 (m, 2h), 4.82 (br, 1h), 4.58 (d, j = 5.5 hz, 3h), 3.95 (s, 3h), 3.303.01 (m, 4h), 2.251.80 (m, 4h), 1.701.30 (m, 17h); c nmr (125 mhz, dmso d 6) 165.7, 165.2, 160.9, 159.8, 156.1, 155.8, 155.7, 155.6, 155.0, 154.4, 145.7, 142.8, 141.0, 140.9, 140.7, 137.3, 136.6, 133.4, 133.3, 130.1, 130.0, 128.9, 128.8, 128.3, 127.7, 117.3, 77.3, 65.1, 64.9, 64.7, 52.0, 48.9, 46.8, 31.8, 31.3, 29.2, 28.9, 28.2, 28.1, 22.8, 22.6; hrms (esi, m + na) calcd for c48h52n10o15na 1031.3511, found 1031.3479 . To a solution of amine 5 (abovementioned) in thf - h2o (1: 1) was added the carboxylic acid 7 (abovementioned), n - methylmorpholine (1.2 ml, 11 mmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (dmt - mm) (3.0 g, 11 mmol), and the mixture was stirred at room temperature for 15 h. to the reaction mixture was added h2o and precipitate was formed . This precipitate was collected with filtration using filter paper, to give 8 as a white solid (3.3 g, 3.2 mmol 89% 2 steps, mp = 200203c). Spectral data for 8: []d = 2.7 (c 1.1, chcl3meoh (1: 1)); h nmr (300 mhz, cdcl3) 8.43 (s, 1h), 8.358.27 (m, 5h), 7.53 (d, j = 8.9 hz, 1h), 7.34 (m, 5h), 5.985.81 (m, 1h), 5.575.44 (m, 2h), 5.30 (d, j = 18 hz, 1h), 5.21 (d, j = 11 hz, 1h), 5.154.97 (m, 2h), 4.82 (br, 1h), 4.58 (d, j = 5.5 hz, 3h), 3.95 (s, 3h), 3.303.01 (m, 4h), 2.251.80 (m, 4h), 1.701.30 (m, 17h); c nmr (125 mhz, dmso d 6) 165.7, 165.2, 160.9, 159.8, 156.1, 155.8, 155.7, 155.6, 155.0, 154.4, 145.7, 142.8, 141.0, 140.9, 140.7, 137.3, 136.6, 133.4, 133.3, 130.1, 130.0, 128.9, 128.8, 128.3, 127.7, 117.3, 77.3, 65.1, 64.9, 64.7, 52.0, 48.9, 46.8, 31.8, 31.3, 29.2, 28.9, 28.2, 28.1, 22.8, 22.6; hrms (esi, m + na) calcd for c48h52n10o15na 1031.3511, found 1031.3479 . Synthesis of 9 to a solution of bis - trioxazole 8 (510 mg, 0.50 mmol) in dmf - thf (1: 5, 30 ml) was added morpholine (440 l, 5.0 mmol) and pd(pph3)4 (29 mg, 0.025 mmol), and the mixture was stirred at room temperature for 1 h. to the reaction mixture was added ether and precipitate was formed . The solid was purified by column chromatography on silica gel (chcl3-meoh = 9: 1) to give 9 (460 mg, 0.50 mmol 99%). Spectral data for 9: []d = 28 (c 1.0, chcl3-meoh (1: 1)); h nmr (300 mhz, cdcl3) 8.43 (s, 1h), 8.358.25 (m, 5h), 7.53 (d, j = 8.9 hz, 1h), 7.33 (m, 5h), 5.565.43 (m, 1h), 5.08 (br, 2h), 4.80 (br, 1h), 4.56 (br, 1h), 4.144.05 (m, 1h), 3.95 (s, 3h), 3.263.04 (m, 4h), 2.251.75 (m, 4h), 1.731.30 (m, 17h); c nmr (125 mhz, dmso d 6) 169.3, 165.2, 160.9, 159.8, 156.1, 155.9, 155.7, 155.6, 154.9, 154.4, 145.7, 142.7, 141.0, 140.9, 140.6, 140.5, 137.3, 136.6, 133.3, 130.0, 129.9, 128.8, 128.5, 128.3, 127.7, 77.3, 65.1, 52.0, 49.4, 46.7, 35.3, 31.3, 29.2, 28.2, 28.1, 22.8, 22.6; hrms (esi, m + na) calcd for c44h48n10o13na 947.3300, found 947.3308 . To a solution of bis - trioxazole 8 (510 mg, 0.50 mmol) in dmf - thf (1: 5, 30 ml) was added morpholine (440 l, 5.0 mmol) and pd(pph3)4 (29 mg, 0.025 mmol), and the mixture was stirred at room temperature for 1 h. to the reaction mixture was added ether and precipitate was formed . The solid was purified by column chromatography on silica gel (chcl3-meoh = 9: 1) to give 9 (460 mg, 0.50 mmol 99%). Spectral data for 9: []d = 28 (c 1.0, chcl3-meoh (1: 1)); h nmr (300 mhz, cdcl3) 8.43 (s, 1h), 8.358.25 (m, 5h), 7.53 (d, j = 8.9 hz, 1h), 7.33 (m, 5h), 5.565.43 (m, 1h), 5.08 (br, 2h), 4.80 (br, 1h), 4.56 (br, 1h), 4.144.05 (m, 1h), 3.95 (s, 3h), 3.263.04 (m, 4h), 2.251.75 (m, 4h), 1.731.30 (m, 17h); c nmr (125 mhz, dmso d 6) 169.3, 165.2, 160.9, 159.8, 156.1, 155.9, 155.7, 155.6, 154.9, 154.4, 145.7, 142.7, 141.0, 140.9, 140.6, 140.5, 137.3, 136.6, 133.3, 130.0, 129.9, 128.8, 128.5, 128.3, 127.7, 77.3, 65.1, 52.0, 49.4, 46.7, 35.3, 31.3, 29.2, 28.2, 28.1, 22.8, 22.6; hrms (esi, m + na) calcd for c44h48n10o13na 947.3300, found 947.3308 . Synthesis of 10to a solution of bis - trioxazole 9 (2.2 g, 2.4 mmol) in thf - h2o (3: 1, 200 ml) was added lithium hydroxide (300 mg, 7.2 mmol), and the mixture was stirred at room temperature for 2 h. to the reaction mixture was added 1n hcl, and the resulting mixture was concentrated in vacuo . To the residual solution in dmf - ch2cl2 (1: 2, 800 ml) was added dmap (1.5 g, 12 mmol), diisopropylethylamine (2.0 ml, 12 mmol), and dppa (2.6 ml, 12 mmol), and the resulting mixture was stirred for 22 h at 90c . To the reaction mixture was added h2o and the organic layer was extracted with ethyl acetate . The extracts were washed with brine, dried over mgso4, filtered, and concentrated in vacuo . The residue was purified by column chromatography on silica gel (ethyl acetate 100%) to give 10 as a white solid (1.7 g, 1.9 mmol 79%, mp = 220225c dec). Spectral data for 10: []d = 12 (c 0.4, chcl3-meoh (1: 1)); h nmr (400 mhz, cdcl3) 8.54 (d, j = 7.3 hz, 2h), 8.258.16 (m, 6h), 7.367.27 (m, 5h), 5.475.37 (m, 2h), 5.05 (br, 2h), 4.89 (br, 1h), 4.59 (br, 1h), 3.222.98 (m, 4h), 2.301.89 (m, 4h), 1.621.18 (m, 17h); c nmr (100 mhz, cdcl3) 164.8, 164.7, 161.2, 159.9, 159.8, 156.3, 156.0, 155.9, 154.6, 141.0, 140.9, 139.1, 138.4, 136.9, 136.8, 136.6, 130.9, 129.6, 128.4, 128.1, 128.0, 79.0, 66.5, 47.8, 47.7, 40.7, 40.3, 34.6, 29.5, 29.2, 28.4, 21.9, 21.8; hrms (esi, m + na) calcd for c43h44n10o12na 915.3038, found 915.2999 . To a solution of bis - trioxazole 9 (2.2 g, 2.4 mmol) in thf - h2o (3: 1, 200 ml) was added lithium hydroxide (300 mg, 7.2 mmol), and the mixture was stirred at room temperature for 2 h. to the reaction mixture was added 1n hcl, and the resulting mixture was concentrated in vacuo . To the residual solution in dmf - ch2cl2 (1: 2, 800 ml) was added dmap (1.5 g, 12 mmol), diisopropylethylamine (2.0 ml, 12 mmol), and dppa (2.6 ml, 12 mmol), and the resulting mixture was stirred for 22 h at 90c . To the reaction mixture the extracts were washed with brine, dried over mgso4, filtered, and concentrated in vacuo . The residue was purified by column chromatography on silica gel (ethyl acetate 100%) to give 10 as a white solid (1.7 g, 1.9 mmol 79%, mp = 220225c dec). Spectral data for 10: []d = 12 (c 0.4, chcl3-meoh (1: 1)); h nmr (400 mhz, cdcl3) 8.54 (d, j = 7.3 hz, 2h), 8.258.16 (m, 6h), 7.367.27 (m, 5h), 5.475.37 (m, 2h), 5.05 (br, 2h), 4.89 (br, 1h), 4.59 (br, 1h), 3.222.98 (m, 4h), 2.301.89 (m, 4h), 1.621.18 (m, 17h); c nmr (100 mhz, cdcl3) 164.8, 164.7, 161.2, 159.9, 159.8, 156.3, 156.0, 155.9, 154.6, 141.0, 140.9, 139.1, 138.4, 136.9, 136.8, 136.6, 130.9, 129.6, 128.4, 128.1, 128.0, 79.0, 66.5, 47.8, 47.7, 40.7, 40.3, 34.6, 29.5, 29.2, 28.4, 21.9, 21.8; hrms (esi, m + na) calcd for c43h44n10o12na 915.3038, found 915.2999 . Synthesis of 11to a solution of macrocyclic bis - amide 10 (200 mg, 220 mol) in meoh (50 ml) was added pd(oh)2/c (80 mg) and the reaction mixture was stirred at room temperature under hydrogen (balloon). After 5 h, the reaction mixture was filtered through a pad of celite, and the filtrates were concentrated in vacuo . To the residual solution in dmf - mecn (1: 1, 4.0 ml) was added diisopropylethylamine (190 l, 1.1 mmol) and adipoyl chloride (16 l, 110 mol), and the mixture was stirred at room temperature for 11 h. the reaction mixture was concentrated in vacuo, and the residue was acidified with 0.1 n hcl and extracted with chcl3 . The organic layer was dried over mgso4, filtrated, and concentrated in vacuo . The residue was purified by column chromatography on silica gel (chcl3acoet meoh = 3: 2: 1) to give 11 (51 mg, 31 mol, 28%). Spectral data for 11: []d = 11 (c 0.95, chcl3-meoh (1: 1)); h nmr (500 mhz, cdcl3) 8.678.48 (m, 4h), 8.278.15 (m, 12h), 6.38 (br, 2h), 5.475.38 (m, 4h), 4.84 (br, 2h), 3.302.98 (m, 8h), 2.151.90 (m, 12h), 1.651.10 (m, 38h); c nmr (125 mhz, cdcl3) 173.0, 164.9, 164.8, 159.8, 159.7, 156.2, 156.1, 156.0, 154.7, 154.6, 141.0, 140.9, 139.5, 139.3, 138.7, 138.6, 136.9, 136.8, 130.8, 129.5, 129.3, 78.9, 47.8, 47.7, 40.3, 38.9, 36.0, 34.5, 34.3, 29.7, 29.5, 28.8, 28.4, 25.1, 21.9, 21.7; hrms (esi, m + na) calcd for c76h82n20o22na 1649.5810, found 1649.5811 . To a solution of macrocyclic bis - amide 10 (200 mg, 220 mol) in meoh (50 ml) was added pd(oh)2/c (80 mg) and the reaction mixture was stirred at room temperature under hydrogen (balloon). After 5 h, the reaction mixture was filtered through a pad of celite, and the filtrates were concentrated in vacuo . To the residual solution in dmf - mecn (1: 1, 4.0 ml) was added diisopropylethylamine (190 l, 1.1 mmol) and adipoyl chloride (16 l, 110 mol), and the mixture was stirred at room temperature for 11 h. the reaction mixture was concentrated in vacuo, and the residue was acidified with 0.1 n hcl and extracted with chcl3 . The organic layer was dried over mgso4, filtrated, and concentrated in vacuo . The residue was purified by column chromatography on silica gel (chcl3acoet meoh = 3: 2: 1) to give 11 (51 mg, 31 mol, 28%). Spectral data for 11: []d = 11 (c 0.95, chcl3-meoh (1: 1)); h nmr (500 mhz, cdcl3) 8.678.48 (m, 4h), 8.278.15 (m, 12h), 6.38 (br, 2h), 5.475.38 (m, 4h), 4.84 (br, 2h), 3.302.98 (m, 8h), 2.151.90 (m, 12h), 1.651.10 (m, 38h); c nmr (125 mhz, cdcl3) 173.0, 164.9, 164.8, 159.8, 159.7, 156.2, 156.1, 156.0, 154.7, 154.6, 141.0, 140.9, 139.5, 139.3, 138.7, 138.6, 136.9, 136.8, 130.8, 129.5, 129.3, 78.9, 47.8, 47.7, 40.3, 38.9, 36.0, 34.5, 34.3, 29.7, 29.5, 28.8, 28.4, 25.1, 21.9, 21.7; hrms (esi, m + na) calcd for c76h82n20o22na 1649.5810, found 1649.5811 . Synthesis of 2a solution of 11 (50 mg, 31 mol) in ch2cl2-tfa (95: 5, 25 ml) was stirred at room temperature for 2 h. the reaction mixture was concentrated in vacuo to give 2 as a white solid (50 mg, 30 mol, 98%, mp = 225230c dec). Spectral data for 2: []d = 72 (c 0.3, chcl3-meoh (1: 1)); h nmr (500 mhz, dmso d 6) 9.159.08 (m, 8h), 8.958.89 (m, 4h), 8.38 (d, j = 6.9 hz, 2h), 8.30 (d, j = 6.9 hz, 1h), 7.807.53 (m, 6h), 5.505.35 (m, 4h), 2.982.89 (m, 4h), 2.782.89 (m, 4h), 2.151.85 (m, 12h), 1.551.00 (m, 20h); c nmr (125 mhz, dmso d 6) 171.7, 164.5, 164.3, 158.8, 158.7, 155.6, 154.5, 142.5, 141.9, 141.8, 141.1, 136.0, 129.8, 129.7, 128.5, 128.4, 47.4, 47.3, 38.6, 38.1, 35.1, 33.1, 28.7, 26.7, 24.9, 21.3, 20.8; hrms (esi, m + h) calcd for c66h67n20o18 1427.4942, found 1427.4961 . A solution of 11 (50 mg, 31 mol) in ch2cl2-tfa (95: 5, 25 ml) was stirred at room temperature for 2 h. the reaction mixture was concentrated in vacuo to give 2 as a white solid (50 mg, 30 mol, 98%, mp = 225230c dec). Spectral data for 2: []d = 72 (c 0.3, chcl3-meoh (1: 1)); h nmr (500 mhz, dmso d 6) 9.159.08 (m, 8h), 8.958.89 (m, 4h), 8.38 (d, j = 6.9 hz, 2h), 8.30 (d, j = 6.9 hz, 1h), 7.807.53 (m, 6h), 5.505.35 (m, 4h), 2.982.89 (m, 4h), 2.782.89 (m, 4h), 2.151.85 (m, 12h), 1.551.00 (m, 20h); c nmr (125 mhz, dmso d 6) 171.7, 164.5, 164.3, 158.8, 158.7, 155.6, 154.5, 142.5, 141.9, 141.8, 141.1, 136.0, 129.8, 129.7, 128.5, 128.4, 47.4, 47.3, 38.6, 38.1, 35.1, 33.1, 28.7, 26.7, 24.9, 21.3, 20.8; hrms (esi, m + h) calcd for c66h67n20o18 1427.4942, found 1427.4961 . Synthesis of 12to a solution of 2 (50 mg, 30 mol) in meoh (5.0 ml) was added amberlyst a-26(oh) ion - exchange resin, and the mixture was stirred for 30 minutes . The resulting mixture was filtered through a cotton with meoh, and the filtrates were concentrated in vacuo . To a residual solution of 2 in dmf (5.0 ml) was added diisopropylethylamine (52 l, 0.31 mmol), hgcl2 (50 mg, 0.18 mmol), and 1,3-bis(tertbutoxycarbonyl)-2-methyl-2-thiopseudourea (66 mg, 0.18 mmol), and the mixture was stirred for 1 h at room temperature . To the reaction mixture was added h2o, and the organic layer was extracted with ethyl acetate . The residue was chromatographed on silica gel (chcl3-ethyl acetate - meoh = 3: 2: 1) to give 12 as a white solid (30 mg, 16 mol, 52%). Spectral data for 12: []d = 3.8 (c 1.4, chcl3-meoh (1: 1)); h nmr (500 mhz, cdcl3) 11.5 (br, 2h), 8.578.48 (m, 4h), 8.308.17 (m, 14h), 6.17 (br, 2h), 5.455.36 (m, 4h), 3.413.32 (m 4h), 3.283.10 (m, 4h), 2.201.88 (m, 12h), 1.651.20 (m, 56h); c nmr (125 mhz, cdcl3) 172.9, 164.8, 164.7, 163.5, 159.9, 159.7, 156.1, 156.0, 154.7, 154.6, 153.2, 141.0, 140.9, 139.3, 139.2, 138.6, 138.5, 136.9, 136.8, 130.8, 129.6, 129.5, 82.9, 79.1, 47.7, 47.6, 40.5, 39.1, 36.0, 34.7, 28.7, 28.6, 28.2, 28.0, 25.0, 22.1, 22.0; hrms (esi, m + na) calcd for c88h102n24o26na 1933.7295, found 1933.7332 . To a solution of 2 (50 mg, 30 mol) in meoh (5.0 ml) was added amberlyst a-26(oh) ion - exchange resin, and the mixture was stirred for 30 minutes . The resulting mixture was filtered through a cotton with meoh, and the filtrates were concentrated in vacuo . To a residual solution of 2 in dmf (5.0 ml) was added diisopropylethylamine (52 l, 0.31 mmol), hgcl2 (50 mg, 0.18 mmol), and 1,3-bis(tertbutoxycarbonyl)-2-methyl-2-thiopseudourea (66 mg, 0.18 mmol), and the mixture was stirred for 1 h at room temperature . To the reaction mixture was added h2o, and the organic layer was extracted with ethyl acetate . The residue was chromatographed on silica gel (chcl3-ethyl acetate - meoh = 3: 2: 1) to give 12 as a white solid (30 mg, 16 mol, 52%). Spectral data for 12: []d = 3.8 (c 1.4, chcl3-meoh (1: 1)); h nmr (500 mhz, cdcl3) 11.5 (br, 2h), 8.578.48 (m, 4h), 8.308.17 (m, 14h), 6.17 (br, 2h), 5.455.36 (m, 4h), 3.413.32 (m 4h), 3.283.10 (m, 4h), 2.201.88 (m, 12h), 1.651.20 (m, 56h); c nmr (125 mhz, cdcl3) 172.9, 164.8, 164.7, 163.5, 159.9, 159.7, 156.1, 156.0, 154.7, 154.6, 153.2, 141.0, 140.9, 139.3, 139.2, 138.6, 138.5, 136.9, 136.8, 130.8, 129.6, 129.5, 82.9, 79.1, 47.7, 47.6, 40.5, 39.1, 36.0, 34.7, 28.7, 28.6, 28.2, 28.0, 25.0, 22.1, 22.0; hrms (esi, m + na) calcd for c88h102n24o26na 1933.7295, found 1933.7332 . Synthesis of 3a solution of 12 (29 mg, 31 mol) in ch2cl2-tfa (3: 1, 2.0 ml) was stirred at room temperature for 2 h. to the reaction mixture was added ether and precipitate was formed . This precipitate was collected with filtration using filter paper, to give 3 as a white solid (20 mg, 12 mol, 80%, mp = 220225c dec). Spectral data for 3: []d = 18 (c 0.75, chcl3-meoh (1: 1)); h nmr (400 mhz, dmso d 6) 9.149.08 (m, 8h), 8.948.90 (m, 4h), 8.37 (d, j = 7.3 hz, 1h), 8.32 (d, j = 7.3 hz, 1h), 7.757.69 (m, 2h), 7.517.45 (m, 2h), 5.485.37 (m, 4h), 3.082.90 (m, 8h), 2.151.84 (m, 12h), 1.501.00 (m, 20h); c nmr (125 mhz, dmso d 6) 171.7, 164.5, 164.4, 158.8, 158.7, 156.7, 155.7, 155.6, 154.5, 142.5, 141.8, 141.1, 136.0, 129.8, 129.7, 128.5, 128.4, 47.4, 40.5, 38.1, 35.1, 33.4, 33.3, 28.7, 28.2, 24.9, 21.3, 21.0; hrms (esi, m + h) calcd for c68h71n24o18 1511.5378, found 1511.5368 . A solution of 12 (29 mg, 31 mol) in ch2cl2-tfa (3: 1, 2.0 ml) was stirred at room temperature for 2 h. to the reaction mixture was added ether and precipitate was formed . This precipitate was collected with filtration using filter paper, to give 3 as a white solid (20 mg, 12 mol, 80%, mp = 220225c dec). Spectral data for 3: []d = 18 (c 0.75, chcl3-meoh (1: 1)); h nmr (400 mhz, dmso d 6) 9.149.08 (m, 8h), 8.948.90 (m, 4h), 8.37 (d, j = 7.3 hz, 1h), 8.32 (d, j = 7.3 hz, 1h), 7.757.69 (m, 2h), 7.517.45 (m, 2h), 5.485.37 (m, 4h), 3.082.90 (m, 8h), 2.151.84 (m, 12h), 1.501.00 (m, 20h); c nmr (125 mhz, dmso d 6) 171.7, 164.5, 164.4, 158.8, 158.7, 156.7, 155.7, 155.6, 154.5, 142.5, 141.8, 141.1, 136.0, 129.8, 129.7, 128.5, 128.4, 47.4, 40.5, 38.1, 35.1, 33.4, 33.3, 28.7, 28.2, 24.9, 21.3, 21.0; hrms (esi, m + h) calcd for c68h71n24o18 1511.5378, found 1511.5368 . The dual fluorescently labeled oligonucleotides flu - telo21 5-fam-[ggg(ttaggg)3]-tamra-3 and flu - ds26 5-fam-[(ta)2gc(ta)2t6(ta)2gc(ta)2]-tamra-3 were used in this protocol . The donor fluorophore was 6-carboxyfluorescein, fam, and the acceptor fluorophore was 6-carboxytetramethylrhodamine, tamra . The oligonucleotides were initially dissolved as a 100 m stock solution in milliq water; further dilutions were carried out in 60 mm potassium cacodylate buffer (ph 7.4). Dual - labeled dna was annealed at a concentration of 400 nm by heating at 94c for 5 minutes followed by cooling to room temperature . We added the different concentrations of ligands into different samples, using a total reaction volume of 40 l, with 200 nm of labelled oligonucleotide . Following experiments should keep the temperature procedure in real - time pcr and procedure was finished as following: 25c for 5 minutes, then a stepwise increase of 1c every minute from 25c to reach 99c . During the procedures, we measured the fam after each stepwise . The 10 m oligonucleotide of telo24: ([ttaggg]4) was dissolved in tris - hcl buffer (50 mm, ph 7.0) and the solution was heated to 90c for 5 minutes, then slowly cooled to 25c . G4 ligands were diluted from 10 mm stock solutions to give a concentration of 1 mm with water and added into the oligonucleotide samples at 50 m (the 10 mm stock solutions of 2 and 3 were made in dmso). Esi - mass spectra were recorded in a negative - ion mode with jeol jms - t100x spectrometer . The direct - infusion flow rate was 5.0 l min . All experiments were performed in 20 mm nh4oac containing 10 m of telo24 and 40 m of 2 and 3 . The 6otd dimers 2 and 3 were synthesized by using the sequences as shown in scheme 1 . Trioxazoles 4 and 6 were synthesized starting with l - serine and l - lysine, respectively by using the previously reported procedure [29, 30, 3638]. The cbz group of 4 was removed by treatment with hydrogen in the presence of pd(oh)2/c to give amine 5 . Hydrolysis of the ester group in 6 with lithium hydroxide followed by coupling of the resulting acid with amine 5 using dmt - mm gave the bis - trioxazole amide 8 . Cleavage of the allyloxycarbonyl group in 8 and hydrolysis of the ester group produced an amino acid, which was subjected to macrocyclization under high dilution conditions (5 mm) to give 6otd 10 . The cbz group in 10 was removed with hydrogen in the presence of pd(oh)2/c to give corresponding amine . The procedure for synthesis of dimer 11 involved coupling of the amine with adipoyl chloride . Bis - amine 2 was obtained by removal of the boc group of 11 with tfa . Preparation of the guanidine derivative 3 was carried out by guanidination of the amine moiety in 2 by using 1,3-bis(tert - butoxycarbonyl)-2-methyl-2-thiopseudourea followed by deprotection of boc group with tfa . With the desired 6otd dimers 2 and 3 in hand, their mode of interaction with the telomeric dna (telo24) firstly, conformational changes of telo24, induced by these substances were evaluated using circular dichroism (cd) spectroscopy . Upon treatment of telo24 with 6otd dimers 2 and 3 (50 m) in the presence of potassium chloride (100 mm), the mixed - type structure induced by potassium cation (solid line in figure 2) is transformed to a typical antiparallel - type g4 structure (dashed line in figure 2) [28, 40]. The binding stoichiometries of the complexes formed between the telo24 and ligands and 3 (molar ratio = 1: 4) were determined by using esi - mass spectrometric analysis [41, 42]. In both cases, only mass peaks that correspond to 1: 1 complexes of both 2 and 3 with telo24 were observed (at the 7-, 6- or in the 5-charge states). Since these interaction modes are the same as that of 6otd dimer 1, the newly synthesized 6otd dimers 2 and 3 appear to interact with telo24 through an end stacking mode using two 6otd moieties in a manner similar to that of tms and/or 6otd dimer 1 (figures 4 and 6) [33,] the ability to stabilize the g4 structure of telo24 by 6otd dimers 2 and 3 was evaluated by employing a fluorescence resonance energy transfer -(fret-) based assay [34, 35]. The tm values of labeled oligonucleotide flu - telo21 with 2 and 3 at a concentration of 1 m, which corresponds to 5 equivalents, are 10 and 14c, respectively (figure 5(a) and table 1). Since under the same conditions the tm value for 1 was 12c, among the substances explored to date guanidine 3 is a potent stabilizer of the g4 structure . This stabilization effect is likely caused by the attractive interaction between positively charged guanidinium residue and anionic phosphates backbone of the telomeric g4 . Interactions of the ligands with the duplex form of flu - ds26 were also investigated by using the same protocol (figure 5(b) and table 1). The observation that no differences in the tm values exist in the presence or absence of dimers 2 and suggests that these ligands are selective for the telomere dna sequence . In summary, the efforts described above have led to the design and syntheses of 2 and 3, two novel macrocyclic hexaoxazole dimeric derivatives of 6otd that have amine and guanidine groups in their respective side chains . These compounds, together with 6otd dimer acetate 1, were found to induce a change of the telomeric dna sequence of telo24 into an antiparallel structure through the formation of 1: 1 complexes with the dna . The guanidine functionalized 6otd dimer 3 was determined to have the greatest ability to stabilize the telomere dna sequence . Also, both dimers selectively bind to the telomeric dna sequence and not double - stranded dna . Further studies, aimed at the structural development of 6otd dimers with different linkers, are currently underway.
The act of eating the earth including clay and chalk is neither new nor outdated . Geophagia, the practice of eating earth is associated with religious beliefs, medicinal and dietary purposes . The implication of this act is the exposure to toxic substances and parasites that are present in the ingested earth. [24] one of such geophagic materials is the calabash chalk, consumed largely in west africa as a remedy for morning sickness; hence it is highly patronized by pregnant women. [57] the chalk is found largely in nigeria and other west african communities . It is also found in ethnic stores and markets in the united states of america and the united kingdom . Different names have been ascribed to this chalk: calabar stone in english, la craie or argile in french, mabele by lingala in congo, nzu by igbo and ndom by the efik / ibibio of nigeria. [57] the major component of c. chalk is aluminum silicate hydroxide from the kaolin clay group, with a possible formula al2si2o2(oh)4 . This chalk is available in varieties of forms including powder, molded shapes and blocks . Lead concentration in c. chalk has been reported to be approximately 40 mg / kg . This concentration of lead in the c. chalk is far more than the approved dietary concentration . Other toxic elements including aluminum, silicon, alpha lindane, endrin, and endosulfan 11 have also been reported . Research carried out with the chalk on rats revealed fragmentation of the parenchymal cells, and dilation of sinusoids of the liver due to treatment with the chalk . Another report has it that it alters the normal concentration of hemoglobin, red blood cell counts and erythrocyte sedimentation rate (esr), of female rats . Since there are reports that this chalk contains heavy metals and other toxic substances, the deposition of these substances in the bone tissue may result in bone disorders and poor growth . Lead, a constituent of this chalk can be localized in areas of bone mineralization and growth . It has been reported that lead adversely influences bone development through disruption of mineralization during growth . As there are no clinical studies, it is important therefore to investigate the effect of c. chalk on the morphometry and mineralization of the bone in experimental animals with the aim of relating the findings to human situation, hence this study was conducted . Fourteen young (weanling) wistar rats of both sexes weighing 54 - 72 g, obtained from the animal house, faculty of basic medical sciences, university of uyo, were housed in wooden cages with saw particles as bedding . The animals were handled in accordance with international regulation governing the use of laboratory animals . The rats were fed with normal commercial pellet (vital feed, grand cereal ltd, plateau state, nigeria) and water was allowed ad libitum throughout the period of the experiment . The room temperature of the experimental animal house ranged between 26 - 29c, with 12:12 hour light and dark cycle . The animals were allowed to acclimatize for 7 days before the beginning of the experiment . Group i served as the control group (cg), while group ii served as test group (tg). Non - salted calabash chalk was bought from a local market in ikot omin, calabar, nigeria . 40 g of the chalk powder was dissolved in 1 liter of water to give a suspension which was stirred prior to administration . Animals in cg were treated with 1ml of distilled water, while those in tg were treated with 1ml of c. chalk suspension containing 40 mg / ml of the non - salted c. chalk . Treatment was given in the morning hours of 8 - 9 am and lasted for 28 days . On day 29, the weight of the animals was noted, and the animals were sacrificed by humane killing using chloroform anesthesia . The weight of the femur bones was measured using a digital weighing balance to the nearest 0.01 g . The length was measured from the head to the condyles of the femur, by means of a measuring tape to the nearest 0.1 mm . The sum of the weight of both the right and left femur were taken as the organ weights, and the organ - somatic index of the femur bones in both groups was computed (organ somatic index = organ weight / body weight). The femur bones were digested using analytical grade nitric acid (3 ml per tissue). Acid digest were diluted appropriately with double distilled - deionized water before analysis of the minerals . Bone phosphate (p) concentration was determined using the acid - molybdate method, and the absorbance read at 400 nm . The analysis for zinc (zn) sodium (na), potassium (k) and calcium (ca) were carried out using graphite furnace atomic mass spectrophotometer and absorbance read at 589 nm, 766.5 nm and 422.7 nm respectively . Data were statistically analyzed using standard student t - test and the results presented as mean standard deviation . Data were statistically analyzed using standard student t - test and the results presented as mean standard deviation . On day 6 of administration, the fecal boluses of the test group (tg) animals were softer in consistency than those of the control group (cg) animals . On day 8, the color of the fecal bolus of the tg was found to be brown to milky color, and this fecal color was maintained till the end of the experiment, unlike the cg that had brownish brown color of fecal bolus . The mean initial body weights (g) of the tg animals were significantly lower (p <0.0006) compared with the cg, while the mean final body weights of the tg animals were also significantly lower (p <0.0008) compared with the cg . The mean change in body weight of the animals in the tg and cg were 52.17 g (47%) and 66.29 g (48.6%), respectively [table 1]. The lengths of the femur bones in the tg were slightly shorter in both the right and the left sides compared to the cg . There was also an insignificant decrease in the weight of both the right and left femur bones of the tg when compared to the cg [table 2]. Weight of animals of the control and test groups weight and length of femur bones of the control and test groups the organ - somatic index in the tg and cg was 0.0025 0.008 and 0.0033 0.004 respectively, with a statistically significant difference (p <0.0126). The mean femur bone concentration (mg / l) of zn, p, ca, na, and k were significantly lower (p <0.05) in the tg compared to the cg [table 3]. Calabash chalk has been reported to contain lead, arsenic and persistent organic pollutants among other substances . The mean concentration of lead as reported by dean et al . Was approximately 40 mg / kg which far exceeded the european union recommended safety level for dietary lead by 40 fold . This and other heavy metals present in this chalk are likely to interfere with the normal process of bone formation . In this study, we investigated the effect of the chalk on bone mineralization and other morphological changes in experimental wistar rats . There was 1.6% decrease in body weight change of the test group (tg). This may result from disruption in nutrient absorption by the chalk, as kaolin, its major constituent, has been reported to coat the lining of the gastrointestinal tract . As a result of this, the coating may have prevented the absorption of beneficial nutrients leading to poor nutrition, which ultimately resulted in a limited body weight gain reported in this study . Beside this, poor nutrition can also increase the risk to other adverse health effects . As one of the poisonous substance found in the chalk, lead may have played a significant role in the body weight reduction observed in this investigation . There were no significant change in the length and weight of each of the right and left femur bones of tg compared to cg, and no significant change between the right and left femur bones in all the parameter measures in both groups . This may be due to the significant change in body weight observed in this study . Furthermore, the results of our investigation showed a significant decrease in the bone minerals content in tg, which may be due to the combined effects of lead and other toxic elements present in the c. chalk . Also, the developing skeleton is much more sensitive to toxicity than that of the adult . Lead accumulates and substitutes calcium in bone tissues and the resultant effect is that of disruption of mineralization, alteration of compositional properties and bone formation mechanisms, as well as the gradual depletion of bone minerals. [2123] lead has also been implicated in reduction of bone strength, oxidative stress, reduced zn, na, copper (cu) and iron (fe) in rat bone . And impaired bone mineral density and content . The reduction in bone mineralization could lead to an increase in bone fragility and osteoporosis . Our study is at variance to a report by jamieson et al . According to this report, femoral, the effect of calabash chalk on femur bone morphometry and mineralization of young wistar rats showed that c chalk may alter growth rate, and cause de - mineralization in the femur bone, and hence it may be detrimental to bone growth . As this is a case in rats, a clinical trial could be carried out on humans to determine its effects.
In conventional information theory and cryptography, it is taken for granted that a digital message can always be copied easily, even by someone ignorant of its meaning . Analog messages (e.g. Handwritten signatures) are somewhat harder to copy, but not really infeasible, and digital data can be protected to a considerable extent by interposing a restrictive hardware interface between the data and the outside world (e.g. Smart credit cards); but in both these cases, the difficulty of copying is only technological, not fundamental . However, when elementary quantum systems such as polarized photons are used as the transmission medium, routine copying of messages is no longer possible even in principle . In particular, there are ways of encoding messages so that they can be copied reliably only with the help of certain key information used in forming the message . Quantum coding was first described in [w], along with two applications: making money that is in principle impossible to counterfeit, and multiplexing two or three messages in such a way that only one can be read . More recently [bbbw], quantum coding has been used in conjunction with public key cryptographic techniques to yield several schemes for unforgeable subway tokens . Here we show that quantum coding considerably enhances the usefulness of another standard cryptographic device, the one - time pad . Mathematically, a polarized photon acts like a two - bit read - once memory one of whose bits (k) serves as a read key for the other (m). Querying the memory with the correct k yields the correct value of m. querying with the wrong k yields a random bit instead of m, and in either case querying resets the memory so that subsequent queries yield no new information . Even after a query, it is generally impossible to infer the initial state of either bit, because the memory gives no indication of whether its response was the correct response to the correct key or a random response to the wrong key . Because it represents the behavior of an elementary quantum system, this kind of restricted - access memory should be thought of as a natural information - processing primitive, not as a complex technological device that could probably be circumvented in principle . Ordinarily, when one thinks of a technological restricted - access memory, one has in mind an information - storage device . Photons can also be stored (e.g. Between mirrors, or in a closed optical fiber), but they cannot in practice be stored for very long, and their natural application is in the transmission of information . We thus have a situation in which restricted - access memory, as a storage device, is possible in practice but not in principle via conventional technology, and in principle but not in practice via storage of polarized photons . On the other hand, restricted - access transmissions, which can be read or copied only with the help of a key, are possible both in principle and in practice using polarized photons . Polarized light can be produced by sending ordinary light through a polarizing apparatus such as a polaroid filter or nicol prism . A beam of polarized light is characterized by its polarization axis, which is determined by the orientation of the polarizing apparatus in which the beam originates . Although polarization is a continuous variable, and in principle can be measured as accurately as desired by passing the polarized beam through a second polarizing apparatus, the uncertainty principle forbids measurements on any single photon from revealing more than one bit about the beam s polarization . In particular, if a beam with polarization axis is sent into a polarizer oriented at angle, the individual photons behave dichotomously and probabilistically, being transmitted with probability \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\cos ^2(\alpha - \beta) $$\end{document} and absorbed with the complementary probability \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sin ^2 (\alpha - \beta) $$\end{document}. The photons behave deterministically only when the two axes are parallel (certain transmission) or perpendicular (certain absorption). If the two axes are not perpendicular, so that some photons are transmitted, one might hope to learn additional information about by measuring the transmitted photons again with a polarizer oriented at some third angle; but this is to no avail, because the transmitted photons, in passing through the polarizer, emerge with exactly polarization, having lost all memory of their previous polarization . Any other elementary two - state quantum system, such as a spin-1/2 atom, behaves similarly dichotomously and probabilistically . Another way one might hope to learn more than one bit from a single photon would be not to measure it directly, but rather somehow amplify it into a clone of identically polarized photons, then perform measurements on these; but this hope is also vain, because such cloning can be shown to be inconsistent with the foundations of quantum mechanics [wz]. In order to encode a message bit m into a photon that can be read or copied reliably only with the help of a key bit k, we generate a photon with a selected one of the four polarization directions 0, 45, 90 and 135 degrees . [generating a single photon of known polarization is possible by variation of the einstein - podolsky - rosen setup [bo], in which a decaying atom emits two oppositely polarized photons . By polarizing and counting one photon, the other s presence is assured and its polarization fixed without measuring it directly .] If the key bit is a 0, then the photon is polarized rectilinearly, i.e. 0 or 90 degrees according to whether the message bit is 0 or 1 . If the key bit is a 1, then the photon is polarized diagonally, i.e. 45 or 135 degrees according to the message bit . The quantum encoding\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_k (m)$$\end{document} of a message m by a key k of equal length is the train of photons obtained by applying the above procedure bitwise to m and k. to read a quantum - encoded message with the help of its key, one simply reads each photon with a polarizer oriented so as to cause it to behave deterministically, for example, reading the rectilinear photons with a 0-degree polarizer and the diagonal photons with a 45-degree polarizer . An attempt to read a photon with the wrong key causes it to behave randomly, losing its stored information . For example, if a 45- or 135-degree photon is read with a 0-degree polarizer, it will be transmitted with 50 per cent probability in either case, and all evidence of its original polarization will be lost . Suppose an eavesdropper intercepts and attempts to read a quantum transmission \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_k(m)$$\end{document} without being detected . Consider first the case in which the message m and key k are both random . Not knowing k, the eavesdropper makes the wrong measurement on half the photons, and thus obtains a message \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m'$$\end{document} differing from m in 1/4 of its bit positions (of course the eavesdropper does not know which ones). Having destroyed the original transmission by reading it, the eavesdropper must now, in order to remain undetected, inject a forged transmission designed to approximate the intercepted one as well as possible . Not knowing which measurements are wrong, the eavesdropper s best strategy is to produce a new train of photons in agreement with the results of the measurements, as if they had all been right . Half of the photons in such a forged transmission will be correct; the other half have wrong key values (i.e. Will be diagonal when they should be rectilinear, or vice versa), and when subsequently measured with the correct key by the intended receiver, these will give wrong answers half the time . Thus the error probability is 1/4 per bit, both for reading the quantum transmission without knowing its key, and for having a forged replacement agree with what the original message would have said when decoded by the intended receiver . Of course, if the intended receiver knew only k but had no prior knowledge of m, the eavesdropping would still1 go undetected, since a random message with random errors still looks random . Quantum money [w] corresponds to the case where the intended receiver (the bank) has perfect knowledge of both m and k, while the counterfeiter knows neither . The usual message m sent over communication channels is intermediate between these extremes: the receiver has partial prior knowledge of it (e.g. Expecting it to be in english). Simply encoding an arbitrary message m with a random quantum key k has two disadvantages: 1) if the message is too random the receiver wo nt be able to detect eavesdropping, for the reason mentioned above; 2) if the message is too redundant (e.g. English), eavesdropping will be detected, but by then the eavesdropper will have gained significant information about the message, perhaps even enough to decrypt it uniquely, because eavesdropping induces errors in only 1/4 of the bits . (in this respect quantum coding differs from ordinary one - time pad encryption, where ignorance of the key prevents the eavesdropper from learning anything about the encrypted message,2 though of course it can be freely copied .) The trick is to make the message redundant with an error - detecting code \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m \rightarrow e(m)$$\end{document}, then hide the redundancy from the eavesdropper by an ordinary one - time pad j, before applying quantum coding . For any error - detecting code e (assumed known to the eavesdropper) let the strongquantum code\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s^e$$\end{document} be defined as follows: let j and k be two random key strings of length \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\|e(m)\|$$\end{document} not known to the eavesdropper.3 then the strong quantum encoding\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s^e_{j, k}(m)$$\end{document} of message m is the train of photons \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$q_k(j {\text {xor}} e(m))$$\end{document}. It is obvious (because of the one - time pad j) that the eavesdropper can learn nothing about m from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s^e_{j, k}(m)$$\end{document}. Moreover, for suitable error - correcting codes,4 eavesdropping incurs a high risk of being detected . Even the rudimentary code of repeating the message twice \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)=mm$$\end{document} suffices to detect eavesdropping with probability at least \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1 - 0.79^k$$\end{document} when k photons have been intercepted, quite close to the optimum \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1 - 0.75^k$$\end{document} implied by the independent, probabilistic nature of eavesdropping - induced errors . Although the simple code \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)=mm$$\end{document} is nearly optimal for eavesdropping - detection, a more complex code would be preferable for another reason: the detection of deliberate message alteration . Although randomly quantum - coded photons cannot be read reliably, they can be altered reliably . For example, the polarization axis of a photon can be rotated by 90 degrees, without measuring or otherwise disturbing it, by passing the photon through an appropriate sequence of mirrors (or, more mysteriously, through a sugar solution). If this manipulation were applied to the first and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(n+1)$$\end{document}st photons of a \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2n$$\end{document}-photon transmission coded as above, both would be altered with certainty in such a way as to induce an undetected alteration in the message . A more complex error - detecting code, e.g. Concatenating mm with a check sum of the addresses of the ones in m, would make such alterations unlikely to escape detection . In the next section, where quantum transmissions are used to carry key information for future transmissions, it will be necessary to use an error - correcting code5 that provides some diffusion, in the sense of making each bit of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)$$\end{document} depend on many bits of m. this prevents the eavesdropper who has luckily guessed a few bits of the present key from thereby efficiently inferring any bits of future keys . Finally, in section 5, we will need a code e that corrects errors as well as detecting them, to make up for photons that arrive at the receiver but fail to be detected . We consider a situation in which two users of an insecure communications channel, who initially share a finite secret key, wish to communicate secretly as long as they can . In a classical setting, where eavesdropping is undetectable in principle, they must assume that all their communications are being listened to, and the volume of safe communication is only linear in the size of the key, unless they resort to pseudorandom key - expansion schemes [bm, y], which are at best (assuming \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {p} \ne \text {np}$$\end{document}) only computationally secure . We show that by strongly quantum - coding their messages with suitable error - detecting codes, the sender and receiver can safely reuse the same \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} keys indefinitely until an eavesdrop is detected . There is an exponentially small chance (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$o(2^{-\|k\|})$$\end{document}) that the eavesdropper, having guessed the entire quantum key \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} correctly, will be able to eavesdrop on all the transmissions without detection and go on to break the reused \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document} key in the usual manner, as well as a moderate chance for the eavesdropper to learn a few bits of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} key correctly and go on to intercept and decrypt a few bits of each message; but these risks do not increase with the number of times an apparently secure key is reused .) An eavesdropper who tampers with or suppresses messages will also be detected with high probability, as will one who injects false messages . When an eavesdrop is detected, the sender and receiver can go on communicating with only slightly diminished safety by replacing their compromised keys by fresh random information sent over the channel in previous uncompromised transmissions . With high probability they will be able to continue communicating in this fashion for an exponential (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{o(\|k\|)}$$\end{document}) number of key changes,6 unless the eavesdrops become so frequent before then that they are forced to use up key information faster than they can replace it, in which case they will (with high probability) be able to cease communication before any of their transmissions have become uniquely decodable by the eavesdropper . Because the replacement keys are truly random, rather than being pseudorandomly computed from an original seed, the security of the scheme would not be reduced by allowing the eavesdropper unlimited computing power . The scheme does incorporate one technologically unrealistic assumption, viz . That photons can be detected with perfect efficiency (cf . Section 5, where this assumption is not made). The ability to send many messages without loss of security (when no eavesdropping is detected) follows from the exponential decline of the probability of escaping detection with the number of distinct bit positions ever subjected to eavesdropping, whether these bit positions are listened to all at once, or a few at a time over the course of many transmissions . For this reason, a quantum channel could even be used to safely send arbitrarily many copies of the same strongly coded transmission, without the eavesdropper being able to forge it accurately, provided the copies were sent one at a time, each only on confirmation that the preceding one had apparently not been listened to . By contrast, if many identical transmissions were sent all at once, the eavesdropper could intercept them all, reliably determine each polarization by multiple measurements, and then escape detection by forging many trains of photons with the now known sequence of polarizations . In order to be sure that no key is reused after a detected eavesdropping, the two communicating parties must alternate strictly in their use of each key . Otherwise the eavesdropper could, for example, intercept and absorb a message from a without forwarding it to b and then wait for b to use the same key on a subsequent message . The effect of absorbing a message is thus the same as that of spoiling it through eavesdropping: neither party reuses the key with which it was sent . If the initial body of shared key information included several keys reserved for first use by a and several for first use by b, the parties could alternate in the use of each key without strictly alternating transmissions . Of course if multiple keys were in use, and particularly if some transmissions were being absorbed by the eavesdropper, the communicating parties would have to prefix each quantum transmission by a (cleartext) indication of which key it was encoded with, to avoid reading a message with the wrong quantum key and spoiling it . The ability to change keys without serious loss of security depends on using a somewhat diffusive error - detecting code when new key information is transmitted . With the simple non - diffusive code \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)=mm$$\end{document}, an eavesdropper who by good luck has correctly guessed the first and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(n+1)$$\end{document}st bits of the current \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} keys will know what measurements to make to reliably read and forge the corresponding bits of a fresh pair of random keys \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j'$$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k'$$\end{document} when these are sent through the channel in four transmissions strongly encoded by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document}; as well as confirming, by the consistency of decoding of the error - detecting code, that the guessed bits of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} are indeed correct . Subsequent lucky guessing on further generations of keys (along with unlucky guessing causing some keys to be rejected due to detected eavesdropping) could be used to discover additional bits until, in linear time, some pair of keys \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j"$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k"$$\end{document} became entirely known . To delay this collapse for an expected exponential number7 of key changes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{o(n)}$$\end{document}, it suffices to use an error - detecting code that diffuses information about each bit of its argument \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m$$\end{document} among many bits of its value \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)$$\end{document}; so that knowledge, say, of any \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n/4$$\end{document} bits of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)$$\end{document} reveals little or nothing about any bit of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m$$\end{document}. Many error - detecting codes have this property, e.g. A random mapping from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n$$\end{document}-bit strings to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2n$$\end{document}-bit strings, or the linear code obtained by mod-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2$$\end{document} multiplying \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$mm$$\end{document} by an appropriate nonsingular matrix . With a diffusive code, knowledge of a few bits of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} would not enable the eavesdropper to make reliable measurements of any bits of the replacement keys \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j'$$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k'$$\end{document}. Although visible light photons can be polarized with nearly perfect efficiency (e.g. A nicol prism can split a beam into two beams, very nearly perfectly polarized at right angles to each other, whose total intensity is scarcely less than that of the incoming beam), and transmitted with nearly perfect efficiency (in a vacuum the only significant losses are due to diffraction, and these can be made negligible by using a beam diameter considerably greater than the square root of the product of the transmission distance and the wavelength of light), current technology allows them to be detected with only about thirty per cent efficiency.8 fortunately, the scheme of the preceding section can be modified to accommodate finite detector efficiency, at the cost of using a more complicated error - correcting code \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m \rightarrow e(m)$$\end{document} in place of the error - detecting code, and a more complicated criterion for key rejection than the detection of a single error on decoding \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)$$\end{document}. Somewhat surprisingly, the modified scheme remains secure against an eavesdropper with a more efficient, or even perfectly efficient, photon detector . The volume of safe communication for this scheme is more than linear, but may be less than exponential, in the initial key size . The main modification is to use standard faint pulses of polarized light instead of single photons, each pulse being of such a size that when it is sent into a detector of the given efficiency (e.g. 30 per cent), or split into several fainter pulses (e.g. By a half - silvered mirror or a nicol prism) and sent into several such detectors, the total number of photons detected obeys a poisson distribution of mean 1 . Such a standard faint pulse can easily be produced by filtering a standard bright pulse of polarized light to reduce its intensity by the requisite constant factor . A standard faint pulse of a given polarization resists copying nearly as well as a single photon would . The best strategy for an eavesdropper to copy a faint pulse is to use a half silvered mirror and two nicol prisms to split the incoming pulse into four beams, one of each canonical polarization, and monitor each beam by a photon detector . Most of the time, only one of the detectors will register a photon, and the eavesdropper will be no better off than in the single photon case . Occasionally two or three detectors will register, yielding more information . Only when three detectors register will the pulse s polarization be known unambiguously (e.g. If both diagonal detectors and the vertical detector register, then the pulse must have been vertically polarized). The faint pulse works well because the chance of three detectors responding to the same pulse is only about 2 per cent (for a poisson distribution of mean~1). The other 98 per cent of the time, the eavesdropper does not learn the pulse s polarization unambiguously, and, as with single photons, cannot reliably copy it . Even a technologically advanced eavesdropper, with perfectly efficient photon detectors, could not copy faint pulses reliably . For example, if the advanced eavesdropper uses 100 per cent detectors to analyze a pulse intended for 30 per cent detectors, an average of 3.3 photons will be detected per pulse, but the chance that these will appear in three different beams, and thus reveal the pulse s polarization unambiguously, would still be only about 25 per cent . The converse phenomenon, namely statistical failure to detect even one photon when a pulse arrives, requires that the rejection test be made more complicated . Even if a transmission is not subjected to eavesdropping, about \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1/e$$\end{document} of its light pulses go undetected, due to normal bad luck at the detectors . The rejection test must begin by deciding whether the number of missing light pulses is so great as to raise the suspicion of eavesdropping (a wise eavesdropper now might not bother to forge replacements for the intercepted pulses, but instead let them remain missing, hoping to pass them off as pulses that arrived but were not detected). If the number of missing pulses is not too great, the error - correcting code must reliably restore the data they would have carried, as well as checking for polarization errors, which as before would indicate interception and forgery of some of the pulses . A convolutional code [g] appears most suitable for achieving the desired high efficiency of error - correction in a channel with a large number of erasures (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1/e$$\end{document}). Depending on the purity of polarization available from the nicol prisms, the code could be made to tolerate and correct a small number of polarization errors, but reject a larger number as evidence of forgery . Since the capacity of a binary channel with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1/e$$\end{document} erasure probability is 0.632, a four - fold expansion in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e(m)$$\end{document} offers ample room for efficient error detection and correction . This in turn means that eight transmissions, each containing n fresh key bits, would have to be accepted to replace the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$8n$$\end{document} bits sacrificed in a rejection . The most problematical aspect of the modified scheme is the decision of when to reject a transmission and change keys . By contrast with the scheme of the previous section, it is now necessary to change keys periodically (at least every \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n^{1/2}$$\end{document} transmissions) even in the absence of any evidence of eavesdropping, in order to prevent an eavesdropper from intercepting all of the bit positions, a few at a time, over the course of many apparently safe transmissions . The expected number of safe key changes has not been worked out, but it is not implausible that it is still exponential in the key size.9
A cross - sectional and longitudinal study was conducted that included 22 medical clinics (i.e., general practitioners) or general / university - affiliated hospitals from different areas in japan, using the same software (codic) to compile electronic medical records, as a working study group, the japan diabetes clinical data management (jddm) study (9). A detailed description of the study has been published previously (9,10). All consecutive patients with type 2 diabetes who visited each clinic / hospital between 2000 and 2009 and whose diabetes was diagnosed before 2009 were included (n = 45,876). In total, 4,798 drug - naive patients were recruited on their first visits between 2000 and 2009 from across japan . All patients met the japan diabetes association criteria for type 2 diabetes (11). All case participants had their height, weight, hba1c, blood pressure (bp), and lipids measured . Bmi was calculated as weight (kg) divided by height squared (m). Waist circumference (wc) was assessed at the top of the iliac crest at the end of a normal expiration (12). The jddm protocol, which is in accordance with the declaration of helsinki, received ethical approval from the institutional review boards of all of the participating institutions and was undertaken in accordance with the ethical guidelines for clinical studies of the japanese ministry of health, labor, and welfare . The morning after an overnight fast, venous blood was sampled for the baseline measurements of the hba1c level and plasma concentrations of glucose, ldl cholesterol, hdl cholesterol, triglycerides (tgs), creatinine, and insulin . Hba1c, expressed in national glycohemoglobin standardization program units, was measured by high - performance liquid chromatography . Plasma total cholesterol, hdl cholesterol, and tgs were assessed with standard enzymatic spectrophotometric techniques . (13), except when tgs exceeded 400 mg / dl (in that case, data were treated as missing). Albumin concentrations in random spot urine samples were determined by turbidimetric immunoassay, and creatinine levels were determined by the enzymatic method . The abbott imx insulin assay (dainabot; abbott laboratories, tokyo, japan) is used to quantitatively measure insulin at bml, inc . This assay shows no cross - reactivity with proinsulin (<0.005%). Within the assay, for every year, the coefficients of variation at mean values of 59.6 and 873.8 the following three mrfs were evaluated as defined by the revised national cholesterol education program criteria (14) and the world health organization criteria for asia (15): 1) wc 80 cm (men) or 75 cm (women); 2) systolic bp 130 mmhg, diastolic bp 85 mmhg, or antihypertensive medications; and 3) hdl cholesterol <1.04 mmol / l, tgs 1.68 mmol / l, or lipid medications . Sex, age, bmi, wc, mean bp, hba1c, fasting insulin, tgs, ldl cholesterol, hdl cholesterol, and microalbuminuria were compared by five consecutive 2-year periods from 2000 to 2009 using one - way anova and the bonferroni post hoc test . Statistical analyses included the unpaired student t test, one - way anova, the test for categorical variables, univariate linear correlations, and ancova . Multiple linear regression analysis was used to assess variables that were independently associated with variations in fasting insulin levels . Both fasting insulin and bmi differences over this 10-year term were assessed using ancova to adjust for potential confounders (sex, age, and fasting plasma glucose [fpg]). The morning after an overnight fast, venous blood was sampled for the baseline measurements of the hba1c level and plasma concentrations of glucose, ldl cholesterol, hdl cholesterol, triglycerides (tgs), creatinine, and insulin . Hba1c, expressed in national glycohemoglobin standardization program units, was measured by high - performance liquid chromatography . Plasma total cholesterol, hdl cholesterol, and tgs were assessed with standard enzymatic spectrophotometric techniques . (13), except when tgs exceeded 400 mg / dl (in that case, data were treated as missing). Albumin concentrations in random spot urine samples were determined by turbidimetric immunoassay, and creatinine levels were determined by the enzymatic method . The abbott imx insulin assay (dainabot; abbott laboratories, tokyo, japan) is used to quantitatively measure insulin at bml, inc . This assay shows no cross - reactivity with proinsulin (<0.005%). Within the assay, for every year, the coefficients of variation at mean values of 59.6 and 873.8 pmol / l were 4 and 2.5%, respectively . Between assays, the coefficients of variation at mean values of 59.6 and 872.4 the following three mrfs were evaluated as defined by the revised national cholesterol education program criteria (14) and the world health organization criteria for asia (15): 1) wc 80 cm (men) or 75 cm (women); 2) systolic bp 130 mmhg, diastolic bp 85 mmhg, or antihypertensive medications; and 3) hdl cholesterol <1.04 mmol / l, tgs 1.68 mmol / l, or lipid medications . Sex, age, bmi, wc, mean bp, hba1c, fasting insulin, tgs, ldl cholesterol, hdl cholesterol, and microalbuminuria were compared by five consecutive 2-year periods from 2000 to 2009 using one - way anova and the bonferroni post hoc test . Statistical analyses included the unpaired student t test, one - way anova, the test for categorical variables, univariate linear correlations, and ancova . Multiple linear regression analysis was used to assess variables that were independently associated with variations in fasting insulin levels . Both fasting insulin and bmi differences over this 10-year term were assessed using ancova to adjust for potential confounders (sex, age, and fasting plasma glucose [fpg]). Comparisons between groups were analyzed for five consecutive 2-year periods versus each variable value in 20002001, when observation began . Sex, age, mean bp, and the prevalence of mrfs (% wc, hypertension, and dyslipidemia) did not differ significantly among year groups . Waist circumference (cm) and bmi were significantly different in the 20062007 and 20082009 groups . Fpg, hba1c, fasting insulin, and tgs were significantly different in each group in 2002 and thereafter . Compared with 20002001, the bmi increased significantly in 2006 and later, but the difference was slight . Blood pressure did not change during the 10-year period . For lipids, compared with 20002001, tgs increased in 2002 and thereafter . As a visual representation of this study, profile plots were created showing the estimated marginal means for the fasting insulin levels / bmi values in each of the 2-year groups (fig . Further analysis using ancova adjusted for age and fpg showed differences among year groups, with an increase in bmi and a decrease in fasting insulin (p <0.001). In each year group, bmi and fasting insulin values in females were higher than in males (p <0.001). Multiple linear regression analysis was performed to identify factors affecting fasting insulin levels (table 2). Factors that significantly affected the fasting insulin value, in order of greater, were wc, bmi, year group, mean bp, hdl cholesterol, tgs, and microalbuminuria . The factors most affecting fasting insulin were wc and bmi, but even after correction for sex, age, and hba1c, the values decreased during the 10-year period . There was also a highly linear relationship between wc and bmi (r = 0.87, p <0.001). Patients' characteristics for each 2-year group estimated marginal mean for ancova results among year groups for fasting insulin (a) and bmi (b). Covariates in each model were evaluated based on age = 56.7 years and fpg = 9.1 pmol / l . In this 10-year study of drug - naive japanese diabetic patients evaluated at their first clinic / hospital visit, fasting insulin levels were found to decrease over time, with wc and bmi being the most important factors after corrections . From 2000 to 2009, when the current study was conducted, a diabetes status survey in japan also estimated an increase in the number of diabetic patients, from 6.9 to 12.5 million (3). It is clear that, in the last 10 years, the insulin secretion ability of japanese individuals has gradually decreased each year . The reason for this is the following changes that were observed in mrfs that play a role in insulin resistance . As mrfs, in the current study, the relationships with wc, bmi, bp, lipids, and microalbuminuria were examined . The factors most involved with the decrease in fasting insulin levels were wc and bmi . Fasting insulin levels were affected by age, sex, and hba1c . Even after adjustment for these, based on our study, the role of mrfs (wc, bmi, bp, lipids, and microalbuminuria) seems important in the pathogenesis of type 2 diabetes in japanese individuals . The asia - pacific region has been considered to be the major site of a rapidly emerging epidemic of diabetes (16), and with its large populations, it is of prime importance for the epidemiology of diabetes . Approximately 13.5% of the japanese population now has either type 2 diabetes or impaired glucose tolerance (17). Furthermore, it is thought that westernization of the lifestyle and an increased percentage of fat in the diet play a role in increasing insulin resistance (18). In asian populations, the -cells may lose their ability to compensate for the decrease in insulin sensitivity seen with the development of central adiposity . Loss of -cell function has been demonstrated to appear before the development of the obesity - induced decreased insulin sensitivity among subpopulations of japanese and japanese americans who develop type 2 diabetes (19,20). The japanese have a higher prevalence of polymorphisms for at least three genes that code for proteins thought to play key roles in lipid and glucose metabolism: the 3-adrenergic receptor, the peroxisome proliferator activated receptor, and calpain-10 (18,21). The interaction between changes in lifestyle and the thrifty genotype characteristic of many japanese people may play a significant role in the increasing prevalence of diabetes and associated cardiovascular risk in this population . Although this type of genetic background has not changed, compared with 30 years ago, dietary habits have become westernized with a high - fat diet . Surprisingly, the daily calorie intake has not changed over the last 50 years and remains at ~2,000 kcal . However, 50 years ago, when a traditional japanese diet was consumed, the percentage of fat in the calorie intake was ~7%, but now it is> 27%, a sudden, almost fourfold increase in only 50 years (3). Therefore, an imbalance with the characteristic japanese insulin secretion ability has developed . As a result, the current state of obesity and poor insulin effectiveness has become very significant . Since japanese people with their lower insulin secretion ability cannot completely compensate for this, the prevalence of diabetes is increasing dramatically . In addition, it has been shown that japanese americans have experienced a higher prevalence of type 2 diabetes than in japan . Research conducted in seattle, wa, suggests that lifestyle factors associated with westernization play a role in exacerbating this susceptibility to diabetes (22). Bmi is a strong determinant of insulin resistance, and it is concordant with the evidence that the mean bmis of representative epidemiologic studies of japanese diabetic patients are from 23 to 25 kg / m lower than in the studies of other ethnic populations (23,24). Bmi and wc thresholds vary among ethnicities, and values are lower for asian populations . Local and regional data have shown that the same level of bmi connotes a greater degree of obesity in asians compared with caucasians (25), and that asians are prone to disorders such as diabetes, hypertension, and dyslipidemia at lower levels of bmi than caucasian populations (25). The use of bmi as a measure of body proportion is a limitation because of its inability to provide information on body fat distribution and central adiposity . The current study also indicates that, for japanese, a slight increase in wc / bmi may be as informative with regard to diabetes risk due to decreasing fasting insulin levels . Decreased -cell function and decreased insulin sensitivity are the two major risk factors for the development of type 2 diabetes . The ability of -cells to compensate for a decrease in insulin sensitivity enables these individuals to maintain normal glucose levels . The fundamental pathological sequence leading to type 2 diabetes is presumed to be the development of an obesity - induced decrease in insulin sensitivity followed by hyperglycemia when the -cells can no longer compensate (27). However, it is still unknown whether -cell dysfunction (28), decreased insulin sensitivity (29), or a combination of both defects is the primary abnormality leading to type 2 diabetes (30). Although the risk factors for type 2 diabetes are similar among ethnically diverse populations, there are ethnic differences in insulin sensitivity and -cell function among groups at high risk for type 2 diabetes . Since the study design was cross - sectional, causal relationships cannot be explored . First, as our study included only type 2 diabetic patients in diabetes clinics / hospitals, our findings cannot be generalized to other patients in a typical practice or community population . Also, fasting insulin levels are limited measures of insulin sensitivity and -cell function . Other environmental or lifestyle factors, including dietary fat or fiber, alcohol consumption, physical activity, smoking, and socioeconomic status might be related to the decreasing trend in fasting insulin over the 10-year period . Nevertheless, we have shown that fasting insulin levels in subjects with newly diagnosed diabetes have been decreasing progressively from 2000 to 2009, and this decrease has been associated with an increase in mrfs, particularly wc and bmi . In conclusion, the superimposition of low pancreatic -cell reserve upon a lifestyle background of metabolic parameters appears to result in hyperglycemia and diabetes in japan.
Brucellosis is endemic in low socio - economic countries like india and the prevalent species causing human infections are brucella melitensis and brucella abortus . Contact with the infected animals, consumption of raw milk and ignorance regarding the disease, are the major risk factors . Since clinical manifestations of this disease are protean in nature, laboratory help is a must in the diagnosis . In the laboratory, brucellosis although an array of serological tests is available, rose bengal plate test (rbpt) and serum agglutination test (sat) are the most widely used . The sensitivity of rbpt is considerably high, but its specificity is much lower when used to test individuals residing in an endemic area . In the same way, sat, used to confirm rbpt results, also has limitations of lack of sensitivity as well as specificity . Apart from this, serological testing does not provide direct evidence for the presence of the pathogen, hence, isolation of brucella spp . From the clinical specimen many of the methods have been developed for culturing brucella spp . From blood specimen, including the conventional ruiz - castaneda (castaneda) method, automated systems, lysis concentration (lc) and clot culture . In general, blood culture for brucella is performed by conventional castaneda method where the blood specimen is directly inoculated in the liquid phase of the castaneda medium . Although the results by this method are satisfactory in acute untreated cases, the incubation time required is very long . The isolation rate is markedly reduced in treated cases or sub - acute / chronic brucellosis patients . The earlier reports have shown an increase in isolation rate and decrease in recovery time by adapting lc technique . However, both conventional and lc methods require one more blood sample for culture once the serological test is found positive . As it is difficult to obtain one more blood specimen in ignorant patients, clot culture could be a better option ., an attempt has been made to compare lc, clot culture and conventional culture techniques for the isolation of brucella from blood specimen in an endemic area . Blood culture by all the three techniques was performed in 169 out of 191 patients showing sat titer of 160 international units (iu) after obtaining written consent . Depending on the duration of the disease, patients were divided into three groups as: acute (<8 weeks), sub - acute (8 - 52 weeks) and chronic (> 52 weeks) brucellosis . Patients were considered to have relapse / re - infection if symptoms reappeared within a year after the completion of antibrucellar treatment . Five ml of blood was collected aseptically from patients with the clinical symptoms resembling brucellosis (fever, malaise, joint pain, low backache, headache, and weight loss) in a sterile, plastic screw capped bottle with 6 - 7 glass beads of 2 mm diameter . The specimen was centrifuged at 2000 rpm for 15 min; the serum was separated aseptically and used for serology . Blood sample for conventional and lc culture technique was collected from patients who had significant sat titers (160 iu). About 10 ml of blood was collected in adults and 5 ml in case of children . The specimen was equally distributed and processed for conventional blood culture and lc technique . The blood specimen was inoculated aseptically into the broth phase of castaneda's biphasic medium consisting of brain heart infusion agar and broth with brucella selective supplement (hi - media)., was employed with a minor modification . 5 ml of blood was added to 40 ml of sterile distilled water with 1.5 ml of 4% sodium citrate in adults and 2.5 ml was added to 20 ml of sterile distilled water with 0.75 ml of 4% sodium citrate in children, in a sterile screw caped centrifuge tube . The contents were gently mixed and the tubes were centrifuged at 7,000 rpm for 30 min . The supernatant was discarded and the sediment was inoculated in the castaneda's medium, instead of culture plate the blood clot preserved in the sterile screw capped plastic tube with glass beads after removal of serum was used for this method . The media were incubated at 37c with 10% co2 for a maximum of 30 days . Sub - culturing was done by allowing the blood broth mixture to flow over the solid phase . The day of appearance of the first colony was recorded for comparison of growth rates . The isolates were identified based on colony morphology, gram stain, modified zn staining, co2 requirement, biochemical tests like oxidase and urease, h2s production for 4 days and growth in the presence of basic fuchsin (1:50,000 and 1:100,000) and thionin (1:25,000, 1:50,000 and 1:100,000). Provisional confirmation and bio - typing of the isolate was done by performing slide agglutination test using b. abortus and b. melitensis monospecific antisera (murex biotech). Statistical analysis of the data was performed by graphpad instat software designed by graphpad software, inc . The blood specimen was inoculated aseptically into the broth phase of castaneda's biphasic medium consisting of brain heart infusion agar and broth with brucella selective supplement (hi - media). . 5 ml of blood was added to 40 ml of sterile distilled water with 1.5 ml of 4% sodium citrate in adults and 2.5 ml was added to 20 ml of sterile distilled water with 0.75 ml of 4% sodium citrate in children, in a sterile screw caped centrifuge tube . The contents were gently mixed and the tubes were centrifuged at 7,000 rpm for 30 min . The supernatant was discarded and the sediment was inoculated in the castaneda's medium, instead of culture plate . The blood clot preserved in the sterile screw capped plastic tube with glass beads after removal of serum was used for this method . The media were incubated at 37c with 10% co2 for a maximum of 30 days . Sub - culturing was done by allowing the blood broth mixture to flow over the solid phase . The day of appearance of the first colony was recorded for comparison of growth rates . The isolates were identified based on colony morphology, gram stain, modified zn staining, co2 requirement, biochemical tests like oxidase and urease, h2s production for 4 days and growth in the presence of basic fuchsin (1:50,000 and 1:100,000) and thionin (1:25,000, 1:50,000 and 1:100,000). Provisional confirmation and bio - typing of the isolate was done by performing slide agglutination test using b. abortus and b. melitensis monospecific antisera (murex biotech). Statistical analysis of the data was performed by graphpad instat software designed by graphpad software, inc . Blood culture by all the three techniques was performed in 169 out of 191 patients . Overall blood culture positivity was found to be 24.8%, 43.1% and 34.9% by conventional, lc and clot culture techniques respectively . The isolation rates in patients with acute, sub - acute, chronic brucellosis and relapse / reinfection by various culture techniques are given in table 1 . Culture positivity by various culture techniques in different stages of brucellosis when compared with the conventional technique lc and clot culture techniques yielded growth in significantly more number of cases (p <0.0001). The mean recovery time for conventional technique was 9.61 days (standard deviations [sd] 1.68), whereas with lc and clot culture techniques it was 4.08 (sd 0.87; p <0.0001) and 5.83 (sd 1.37; p <0.0001) respectively . In the acute stage, earliest growth by the conventional technique was seen on the 6 day where as it was on the 3 day with both lc and clot culture techniques . Lc technique showed earlier growth in 96% blood cultures, before the first positive culture by conventional method was noted, whereas clot culture yielded earlier growth in 42.55% of cultures . Maximum incubation time required for culture to be positive by lc, clot culture and conventional technique was 6, 9 and 16 days respectively . After 16 day, no culture was found positive, though the cultures were incubated for 1 month . Despite maximum aseptic precautions taken during collection and processing of the sample, contamination rate was high for clot culture (5.32%) followed by lc (2.95%), may be due to more number of manipulations required for the procedure . In the present study, blood culture positivity was found to be 24.8%, 43.1% and 34.9% and the recovery time was 10, 4 and 6 days by conventional, lc and clot culture techniques respectively . Our findings suggest that lc and clot culture methods are more sensitive and require less time than conventional culture thereby helping in early detection of infection . Among the lc and clot culture techniques, lc was better due to increased isolation rates and decreased recovery time ., the mean recovery time by lc in this study was 4.08 days, which is in agreement with espinosa et al ., but slightly more as compared to the results of 2 and 2.7 days by etemadi et al ., and mantur and mangalgi . In a study done by escamilla et al ., clot culture technique was found to be less sensitive and more labor - intensive when compared with the conventional method . Conversely in our study, clot culture technique was more sensitive and also the recovery time was less when compared with the conventional method ., another advantage of this technique is that need for one more blood sample is alleviated . In a study by espinosa et al ., isolation rate was higher in sub - acute cases than in patients with acute brucellosis by lc technique . Whereas far higher isolation rates in both acute and chronic stages have been reported by mantur and mangalgi . In our study, culture positivity rate was high in patients with acute followed by sub - acute brucellosis and none of the chronic cases grew brucella . As the patients in our study were not relieved of their symptoms over a long time, they switched on to many doctors before being diagnosed as brucellosis cases at our hospital, which could be an attribute for our lower isolation rates . History of minimum 3 - 4 courses of antibiotics was elicited in 116 (68.63%) patients . In general, blood cultures are attempted to confirm the presumptive serological diagnosis . In india, especially in rural areas, due to the misperception that blood once lost is lost forever and blood loss enhances the disease; it is difficult to obtain one more blood specimen for culture in seropositive individuals . In such situations and also in field conditions (camps), wherein obtaining a second sample for culture is difficult, clot culture could be the optimal alternative . Both lysis and clot culture require manipulations, which enhance the chances of laboratory infection and contamination, thereby necessitating strict biosafety and aseptic measures . We propose that for the isolation of brucella from blood specimen, lc method is better than conventional castaneda's method as the isolation rate is high and the recovery time is less . Clot culture is a better option when a second blood sample cannot be obtained for culture . As lysis and clot culture techniques are sensitive, simple and inexpensive and yield earlier results, they can be adapted in the technically and economically backward areas where automated systems are far from reach . However, to prevent laboratory acquired infection, these methods are to be carried out in class ii biosafety cabinet with bsl-3 precautions.
Defining cancer stem cells and their origins is of much controversy, and constitutes a challenged knockout for cell targeting- anticancer drugs . Herein, we put forward a hypothetic model for cancer stem cells initiation from bone marrow stem cells . These later, will differentiate into an ancestral progenitor that activates a memorial program - the black box cassette- that is responsible of abnormal neo - organogenesis in the form of tumors and metastases . To approve this model, we assume that characterizing and investigating the most primitive forms of the bone marrow progenitors is required; both inside their niche and in circulation of cancer patients . Even as tremendous inventions had have ameliorated anticancer therapeutic practices, these pathologies' developmental and progressive mechanisms are still disputed; and inclined a chiefly limiting factor in front of drugs' efficiency . Consequently, several opinions and hypotheses have been formulated to define tumorigenesis and metastasis origins . Johannes muller (1838) and her student rudolph virchow (1821 - 1902) were the first pathologists describing the cellular origin of cancer . Huge plethora of studies has depicted, one by one, cascades of cancer development, to finally draw the cancer stemness theory which is reinforced by several molecular and genomic scrutinies . Because of the important heterogeneity of the characterized cancer stem cells (cscs), which are harbored within different kinds of tumors; it was stubborn to accurately demarcate their origins . In regard of the copious common patterns shared with cscs, we hope to highlight relevant opinions dealing with the possibility that bone marrow progenitors did initiate the cancerous transformation . The cell transformation and progression, from normalcy to malignancy, could be simulated to a honey - bees' colonial development and reproduction . This insect's queen gives naissance to numerous and various individuals, amongst which only rare genetically defined mature females (future queens) will generate new outposts, when and where conditions are favorable . Thus, in certain circumstances, normal cell will transform to a csc through several mechanisms, and acquire a genomic program enabling it to get patterns of " honey- bees' queen " . Several works impressively assembled and described the cellular, molecular and genetic circuitry that is ensued by the transformed cell to result into metastases [4, 5]. They commonly stippled for a repeated cascade of events that permit various capabilities for tumoral cells: self- sufficiency in growth signals, insensitivity to antigrowth signals, escaping to apoptosis, unlimited replicative potential, sustained angiogenesis, tissue invasion, migration and metastasis . Nonetheless, these events are physiological alterations, eroding the multi - cellular organism homeostasis, that are guaranteed by both the csc (intrinsic factors) and the surrounding microenvironment (extrinsic factors). The genetic and epigenetic alterations constitute the major intrinsic factors leading to the disease's development and progression; while extrinsic factors assemble various elements such as immune surveillance depletion; aging and inflammation . These two factors constitute the basic trait for paget' hypothesis: the seed and soil . Seemingly, the reliability between intrinsic and extrinsic elements outlines a rate of knot for csc evolutionary scenario . Thus, it is conceivable that each genomic modification might, reciprocally, match with an adequate environmental variation point . Whether these two sequential events fit one to other, is still an important inquiry to enlighten . It was assumed that, at least, two or more mutational events (genomic modifications) should occur to initiate the primary cancerous transformation; but to raise into metastasis in distant organs several passive and/ or active modifications are required, in both cscs' daughters and their surrounding microenvironment . Garraway and sellers (2006) have developed a model of aberrant lineage ontogeny and somatic genetics, in order to explain the chronological evolving of the disease . They proposed that tumorigenesis and metastasis arose from the activation of an inborn " backward " developmental program of lineage progenitors and their survival genetic programs . Such system might generate an ancestral form of stem cells that aberrantly grow and invade into the body, like as it occurred during organogenesis . Hold inactive in normal cells' memory, this black box cassette is unexpectedly reactivated under some defined conditions, and will turn on a fastidious machinery of organogenesis leading to tumor development and metastases . In conjunction to the cancer stemness theory, this conception could bring simple explanations in oncology . Bone marrow constitute the primarily reservoir of stem cells (bmscs) in the body . When bmscs are in contact with other tissues, they give raise to tissues' neo- genesis . Also, it has been revealed that bmscs acquire tissue morphology and landmarks, by spontaneous fusion with normal cells [15, 16]. Thus, it is plausible to seek for bmscs as the origin of various cancer diseases . Some experimental models of bmscs transplantations into nude or immune- compromised animals prompted relative improving for their transformation into solid tumors [17 - 19]. This pathologic deviation of bmscs could be mediated by the accumulated chromosomal instability, as observed in murine model . Using similar experimental model, zheng and liang (2008), showed that the donating - males' bmscs did not contribute to the induced hepatocellular carcinoma into receptive females . Such debate might be caused by the important conditioning, like the immune system depletion that permits not only tumorigenesis but also any introduced pathogen's growth . So, clinical investigations are likely much relevant to improve our knowledge on the pragmatic release of tumorigenic cells from bmscs . Beyond, the great assays examining the genetic and cluster designating landmarks of cells, bmscs have been identified in the core of tumor, in cancer patients . Reciprocally, cancerous cells were found to circulate and to home into the bone marrow niche . This mutual relocation of both tumorigenic cells and bmscs derived ones rappels the circulatory loops of immune cells . Interestingly, osteosarcomas that could directly originate inside the bone marrow niche, is the most frequent metastasis in childhood . Furthermore, in myeloma patients, there are several chromosomal translocations that occur into osteoclasts' nuclei . According to the authors, these genetic aberrations are hybrids of osteoclasts with myeloma cells . Tumoral cells have been characterized into bone marrow aspirates from non- metastatic, and disease- free breast cancer patients, after systemic recovery; and in earlier stages (i and ii) of the disease [23, 26]. (2011), who summarized that the tumor progression is firmly dependent on bone marrow stromal cells' lineages . The most primitive form of bmscs, the innate bmsc, will replicate and self - renew to generate various normal progenitor lineages (hscs: hematopoietic stem cells, and bmdscs: somatic bone marrow derived stem cells), in order to ensure homeostasis and damaged tissues' repair . Under the influence of various extrinsic and eventually intrinsic factors, these progenitors will systematically lead to different kinds of the known myeloma; diffuse and follicular lymphomas; and distant sarcomas (yellow panels). (2012) showed that a pluripotent stem cell derived from bmscs could re - differentiate into hematopoietic cells . This prompted the possibility that bmscs produces an ancestral form of progenitors that exhibit an active engine dotted for organogenesis: the black box cassette (green panels). These ancestral cells exhibit embryonic - like stem cells properties and could be found both into bone marrow niche and the blood circulation . Ultimately, they will easily enter sequential and spontaneous differentiations to any kind of stromal cells progenitors and migrate to a definite organ where they clone and contribute to tumorigenesis . Perhaps, there will be swaying and switching between the yellow and green pathways, dependently on lineages' nature and the surrounding microenvironment (dotted lines). Perhaps, this might explain the possible reversibility of the metastatic neuro - blastoma observed in childhood . Furthermore, this theoretical scenario of tumor development and metastasis could reveal the mystery of cancers without defined origins . Several investigations evidenced the existence of such memorial program, in various stromal cells, which was expected to be under epigenetic factors' control [33, 34]. So, the consecutive spatio- temporal variation in derived progenitor cells would be subsequent to dna methylation dynamic that is supposed to guide memory in stem cells . Since that, the epigenetic alterations in bmscs will prompt the black box cassette program to viaduct an erroneous organogenesis (figure 1). Our opinion outlines that cancer initiating- cells could derive from bone marrow progenitors . To do so, bmscs are firstly pushed to generate an ancestral form of progenitors that have an activated memorial program - the black box cassette- leading to fastidious tissular neo - genesis . This scenario, did involve few genomic modifications that are needed for the ancestral cell release; whereas tumorigenesis and metastases progression are spontaneously mediated without requirement of further genetic interventions . In this way of thinking, the intra and inter- tumoral cells' heterogeneity and diversity are quietly acquired during both the colonization or at the first transformations leading to various ancestral progenies of stem cells . However, it could be envisaged that they are temporarily expressed by the progenitors as they have instable genomes . To get better comprehension concerning this hypothesis, we suggest that fine - tuning of cells' depicting techniques toward the characterization and isolation of the most primitive forms of cancer patients' bmscs, into both bone marrow niche (aspirates) and the circulation (blood and lymph), is necessary . Thereafter, accurate experimental models will be helpful to find the relationships between these cells and tumorigenesis and metastasis.
The successful isolation of individual layers of graphene in 2004 has triggered an intense interest in its unique structural and electronic properties . A very high carrier mobility along with weak spin orbit and hyperfine interactions predestinates graphene as a promising material for spintronics, mainly if magnetic ordering can be introduced . However, due to the delocalized bonding network, ideal graphene is intrinsically nonmagnetic . Therefore, developing effective methods for synthesizing ferromagnetic (fm) graphene with high magnetization is vital for applications in novel spintronic devices combining charge and spin manipulation . A number of factors including atomic vacancies, zigzag edges, sp functionalization, and chemical doping of foreign atoms can induce localized magnetic moments in graphene, which are indispensable for the existence of magnetic ordering . Among these strategies, doping of the graphene lattice with noncarbon atoms has been identified as a promising approach for imprinting magnetic ordering into graphene while preserving / enhancing its electric / optical properties, as desirable for spintronic, optoelectronic, and magnetooptical applications . Substitutional doping of graphene by light elements has recently attracted much attention (see, e.g., refs (1114)). In particular, nitrogen doping of the graphene lattice has been extensively studied, aimed at tuning the graphene electronic features, and hence its physical properties, in order to meet the requirements of a given application . N - doped graphenes have been tested as active materials for li - ion batteries, fuel cells, field - effect transistors, ultra- and supercapacitors, and in the fields of photocatalysis and electrochemical biosensing . Chaban and prezhdo have argued that graphene structures containing 1/3 of nitrogen atoms or less should be stable up to 1000 k provided n having similar atomic size and one additional electron compared to a carbon atom, nitrogen acts as an n - type (electron - donating) dopant by increasing the number of electrons when substituted into the graphene lattice, thus affecting the graphene electric conductivity . The substitution of carbon by nitrogen atoms in the graphene lattice necessarily leads to changes in the electronic density of states, and graphitic nitrogen can provide electrons close to the fermi level of graphene . If the itinerant electrons occupy narrow bands at the fermi level, stoner magnetism can emerge, as recently shown for graphene doped with sulfur . Indeed, it has been theoretically proposed that depending on the concentration and packing geometry of doping nitrogen atoms, it is possible to induce a magnetic response in graphene . However, the physical mechanism governing the emergence of magnetically ordered structures was not discussed . Until now, all the experimental attempts to imprint magnetism into graphene through n - doping have failed . Recently, a detailed theoretical and experimental magnetic study revealed that introduction of pyrrolic nitrogen into the graphene lattice decreases the magnetization values compared to those observed for defective graphene .%) was identified to enhance ferromagnetism in highly oxidized graphene (i.e., graphene oxide) synthesized via hydrothermal reaction . Fm behavior has also been reported in other nitrogen - doped graphene oxide systems . However, n - doped graphene oxides are unsatisfactory because a high amount of oxygen (sp functionalization) is regarded as a dominant source of magnetism, overwhelming the effects of nitrogen doping itself . Moreover, oxygen - containing functional groups drastically reduce the electric conductivity of graphene the main prerequisite for spintronic technologies . Thus, development of fm n - doped graphene with negligible oxygen content and elucidation of the effect of various nitrogen configurations (pyrrolic, pyridinic, graphitic, chemisorbed) remains a key challenge in the fields of nanotechnologies, magnetism, and spintronics . In this work, we first report the emergence of a magnetically ordered state in graphene doped solely with nitrogen .% of nitrogen, the doping - induced paramagnetic centers commenced to interact magnetically, resulting in establishment of fm ordering at temperatures below 69 k. in the fm state, the saturation magnetization reached 1.09 emu / g, which is among the highest values reported so far for doped graphene - based systems . Dft calculations demonstrated that graphitic nitrogen was dominantly responsible for evolution of the magnetically active configurations . In contrast, pyridinic and chemisorbed nitrogen had a considerably lower effect on imprinting the magnetism into graphene . This finding opens possibilities for an extensive research in spintronic and magnetooptical technologies based on graphitic n - doped graphene . Graphite microparticles (215 m, 99.9995%, alfa aesar) were used for all syntheses . Sulfuric acid (98%), nitric acid (68%), potassium chlorate (99%), hydrochloric acid (37%), silver nitrate (99.5%), and barium nitrate (99.5%) were obtained from penta, czech republic . Nitrogen (99.9999% purity) and ammonia (99.9995% purity) were obtained from siad, czech republic . Briefly, sulfuric acid (98%, 87.5 ml) and nitric acid (68%, 27 ml) were added to a reaction flask (pyrex beaker with thermometer) containing a magnetic stir bar . Graphite (5 g) was then added to the mixture under vigorous stirring and, keeping the reaction flask in the ice bath, potassium chlorate (55 g) was slowly added . After a complete dissolution of potassium chlorate, the reaction flask was loosely capped to allow the escape of gas evolved and the mixture was continuously stirred for 96 h at room temperature . The obtained graphene oxide was then redispersed in hcl solution (5%, 3 l) to remove sulfate ions and repeatedly centrifuged and redispersed in deionized water until a negative reaction on chloride and sulfate ions with silver and barium nitrate was achieved . The resulting graphite oxide was dried in a vacuum oven at 50 c for 48 h. nitrogen - doped graphenes were prepared by combined exfoliation and reduction of graphite oxide in an ammonia atmosphere . Briefly, 100 mg of graphite oxide was placed inside a quartz glass capsule connected to a magnetic manipulator and mounted in a horizontal quartz glass reactor . Subsequently, the sample was inserted in the hot zone of the reactor while the ammonia flow was kept at 1000 ml min and the pressure was 100 kpa . The temperature of the sample was held constantly for 12 min at 400, 600 or 800 c . After removal from the hot zone of the reactor, the sample was cooled in an ammonia atmosphere . The gn0.015 sample was prepared at 400 c, gn0.033 sample was prepared at 600 c, and gn0.051 sample was prepared at 800 c; the subscript reflects the level of nitrogen in the respective sample as identified from the analysis of the respective survey x - ray photoelectron spectroscopy patterns .% in all the samples studied . Compared to thermally reduced graphene oxide (trgo), used as a blank undoped sample, a slight increase in the oxygen content can be explained in terms of competing processes of nitrogen incorporation and reduction of graphite oxide . Trgo sample was prepared at 800 c by a similar procedure using only nitrogen (1000 ml min) as an exfoliating atmosphere . The residual metal content in the trgo and n - doped graphene samples was analyzed by inductively coupled plasma mass spectrometry (icp - ms). An exact amount of sample (10 mg) was immersed in concentrated nitric acid (99.999% trace metals basis) and heated for 2 h at 100 c . Afterward, the mixture was transferred into a 10 ml volumetric flask, diluted with water and any undissolved graphene was separated using a 200 nm millipore filter . The measured concentration of metals in the solution was recalculated to the amount in the tested sample (analogically, diluted nitric acid was used as a blank). The atomic percent of c, o, and n, and types of bonds were assessed by x - ray photoelectron spectroscopy (xps), employing a phi 5000 versaprobe ii xps system (physical electronics) equipped with a monochromatic al k source (15 kv, 50 w) with a photon energy of 1486.7 ev . All the xps patterns were measured in a vacuum of 1.4 10 pa and at room temperature (22 c). For high resolution xps patterns, a pass energy of 23.500 ev and step size of 0.200 ev xps patterns were evaluated with a multipak (ulvac, phi, inc .) All binding energy values were referenced to the c 1s peak at 284.80 ev . Raman spectra were acquired using a dxr raman spectroscope (thermo scientific, u.s.a .) The respective sample was first deposited on a glass platform and the excitation laser was focused on its surface . The laser power on the sample was set to 5 mw and the exposition time was 20 s. each measured raman spectrum was an average of 16 experimental microscans . Thermogravimetric analysis (tga) and evolved gas analysis (ega) curves were recorded using a netzsch sta 449c jupiter system with an adapted qms 403c aolos quadrupole mass spectrometer . Tga / ega measurements were performed in an open -al2o3 crucible under an argon atmosphere (80 cm min). A temperature program from 40 to 1000 c with a heating rate of 5 k min was used . Magnetization measurements of the trgo and n - doped graphene samples were performed using a physical property measurement system (ppms) equipped with a vibrating sample magnetometer (vsm) from quantum design, u.s.a . Hysteresis loops were measured over the temperature range from 5 to 300 k and under static external magnetic fields ranging from 50 to + 50 koe . Temperature profiles of the mass magnetic susceptibility, mass, were recorded in a sweep mode over a temperature range from 5 to 300 k in a field of 1 koe after cooling in a field of 1 koe . Magnetization values were corrected assuming the response of the sample holder, sample capsule, and respective pascal constants . Atomistic calculations were performed using a spin - polarized density functional theory (dft) and projected augmented wave potentials (paw) representing atomic cores as implemented in the vienna ab initio simulation package (vasp). Electronic exchange and correlation effects were treated by using the perdew, burke, and ernzerhof (pbe) generalized - gradient approximation (gga) with a plane wave cutoff of 600 ev . Brillouin zone integrations were performed with a 6 6 1 point - centered monkhorst pack k - point mesh per conventional 4 3 rectangular cell (structure and cell optimization). The electronic density of states was calculated using the tetrahedron method with a 21 21 1 k - point mesh . Partitioning of the ground state electronic density into contributions attributed to the different atoms was performed exploiting the bader analysis . Total magnetic moments were calculated from the difference between the number of electrons in occupied majority- and minority - spin states . Local magnetic moments were calculated by projecting the plane - wave components of all the occupied eigenstates onto spherical waves inside an atomic sphere and integrating the resulting local density of states . A full structural optimization was performed using a quasi - newton algorithm until the residual atomic forces were lower than 25 mev . Simultaneously, the electronic and magnetic degrees of freedom were converged to an energy of less than 10 ev . The stability of the reported configurations was analyzed in terms of the formation energy, ef, of the n - doped complexes using the formula given as ef = 1/n[edpd egraph + n(c n)], where edpd and egraph stand for the total energies of the doped and pristine graphene sheet, respectively, c and n are the chemical potentials of c and n atoms, respectively (here, approximated by the atomic energies of c and n), and n is the number of substituted atoms . A positive ef indicates that the doping process is endothermic, although it does not hinder the formation of thermodynamically stable complexes . The adsorption energy (ead) per n atom (n = 1 and 2) on a pristine layer was calculated as the total energy difference between the energy of an adatom - graphene complex, en / graph, a clean graphene layer, egraph, and an isolated n atom in the gas - phase, en, i.e., ead = 1/n(en / graph egraph nen). Similarly, the adsorption energy of on - surface nitrogen on n - doped graphene was calculated as the total energy difference between the energy of a doped graphene with an adatom, en / dpd, a doped graphene sheet, edpd, and a gas - phase n atom, i.e., ead = en / dpd edpd en . To study the role of n - doping on imprinting magnetic features into graphene, three samples differing in nitrogen content were prepared; the level of doping was solely controlled by the temperature, while all other synthetic parameters were kept constant . The trgo sample was synthesized as a reference blank sample for which no source of nitrogen (i.e., ammonia atmosphere) was used during the thermal reduction of graphite oxide . The reduction process was found to be highly efficient, as evident from the very low content of oxygen (1 at .%, see the survey and high - resolution c 1s xps pattern in figure s1a, b in supporting information). The paramagnetic / fm response of graphene evolved due to defects and/or functionalization is of several orders of magnitude lower compared to that of 3d - block metals, such as iron, cobalt, nickel, and manganese . Hence, the results of magnetization measurements may be incorrectly interpreted if these strong magnetic elements are present in the system as a consequence of using reactants containing 3d - block metals and/or the sample handling . Thus, icp - ms was employed to quantify the presence of 3d metal impurities and exclude their effect on the magnetic properties of n - doped graphenes . The total concentration of fe, ni, co, and mn, regarded as the main magnetic impurities in trgo and n - doped graphene samples, was below 10 ppm (see table s1 in supporting information). Taking into account the determined concentrations and magnetic moments of the metal impurities, the total mass of fe, ni, co, and mn was estimated to be of the order of 10 emu g oe at 0 k and in a 1 koe field . As mass values for trgo and n - doped graphene systems reached orders from 10 down to 10 emu g oe in a 1 koe field (see below), the contribution of fe, ni, co, and mn to the samples mass was assumed to be negligible in measurements of temperature evolution of mass and hysteresis loops, thus definitely not overshadowing the magnetic properties of graphene induced solely by nitrogen doping . Doping of graphene with nitrogen was monitored by xps and raman spectroscopy . In survey xps patterns recorded for n - doped graphene samples, peaks belonging to c, n, and o were clearly observed (see figure s2 and table s2 in supporting information). The content of nitrogen was found to increase progressively with the temperature at which the thermal reduction of graphite oxide in the presence of ammonia was conducted (i.e., 400 c, 1.5 at . The presence of nitrogen was further evidenced in the high - resolution c 1s xps profile, which showed the emergence of a peak at a binding energy of around 285.5 ev corresponding to the c n spectral component increased in area with the level of n - doping . A small shift in the maximum of the c n peak witnessed for the three n - doped graphene samples can be explained in terms of impossibility to distinguish differently coordinated nitrogen atoms with carbon atoms in graphene with similar binding energy values in the c 1s domain and the significant overlap of the c n and c o spectral components . High - resolution n 1s xps patterns of n - doped graphene samples (see figure 2 and figure s3 and table s3 in supporting information) showed three distinct peaks corresponding to nitrogen in different configurations inside a graphene lattice or attached covalently to a graphene sheet (see figure 3), i.e., pyridinic nitrogen (at 398.3 to 398.5 ev), graphitic nitrogen (at 401.0 to 401.5 ev), and chemisorbed n / n2 (at 404.5 to 405.5 ev). In contrast, no traces of pyrrolic nitrogen, usually present at 400.0 ev, were observed (compare the high - resolution n 1s xps patterns shown in figure 2 and figure s3 in supporting information with figure 3). The pyridinic and graphitic nitrogen were viewed as nitrogen incorporated inside the graphene lattice, whereas chemisorbed n / n2 was viewed as nitrogen adsorbed as adatoms . The presence of n / n2 was further confirmed by tga and ega techniques with gas electron ionization mass spectrometry, indicating emission of a fragment with m / z = 28 (for n / n2) above 300 c (see figure s4 in supporting information). In addition, as no fragments with m / z = 30 and 48 were detected, the samples were considered free of any no and no2 species, respectively . This agrees with the analysis of the raman spectra of n - doped graphene samples as no raman peaks around 1430 cm, characteristic for no and no2 species, are observed (see figure 1b, d, f). High - resolution c 1s xps patterns of the (a) gn0.015, (c) gn0.033, (e) and gn0.051 sample with bonds indicated . Raman spectra of the (b) gn0.015, (d) gn0.033, and (f) gn0.051 sample with the id / ig ratio indicated . High - resolution n 1s xps pattern of the gn0.051 sample with peaks assigned to differently coordinated nitrogen . Scheme showing different bonding configurations of nitrogen in n - doped graphene and corresponding peaks in a simulated high - resolution n 1s xps pattern . Raman spectra of the gn0.015, gn0.033, gn0.051, and trgo sample are shown in figure 1b, d, f and figure s1c in supporting information . It can be seen that on increasing the level of n - doping, the d - to - g band intensity ratio, id / ig, increased . It is known that if nitrogen enters the graphene lattice, the intensity of the d - band, i d, in the raman spectrum of graphene is enhanced due to defects that emerge upon incorporation of nitrogen into the graphene structure . This provides further evidence that under the synthetic conditions used, accommodation of nitrogen into the graphene lattice was strongly favored over a simple adsorption / addition process . If defects are introduced into graphene, paramagnetic centers emerge that may interact via suitable mediators (i.e., -conduction electrons, overlapping orbitals favoring superexchange mechanism, etc .) To generate long - range magnetic ordering in the 2d lattice . Under such conditions, the magnetic susceptibility of graphene,, involves three contributions, i.e., = dia + para + ferro, where dia is the diamagnetic term including orbital, landau and core diamagnetic contributions, para is the paramagnetic term including noninteracting (isolated) defect - induced paramagnetic centers, pauli paramagnetic contribution from conduction electron and van vleck contribution, and ferro is the fm term describing the magnetic response of interacting defect - induced paramagnetic centers . As expected, pristine trgo behaved in a diamagnetic manner with only a tiny paramagnetic response at low temperatures (see figure s1d in supporting information) due to paramagnetic centers emerging as a result of defects and/or the negligible content of oxygen functionalities, which were most likely at the edges; the profile of mass of trgo well matched the modified curie law, i.e., mass = mass, dia + c / t, where mass, dia is the diamagnetic contribution, c is the curie constant, and t is the temperature . Similarly, the temperature evolution of mass measured for the gn0.015 and gn0.033 sample also obeyed the modified curie law (see figure 4a, b). The number of paramagnetic centers increased upon increasing the level of n - doping, as expected and evidenced by an enhanced paramagnetic curie contribution (see insets in figure 4a, b). However, n - doping at such levels did not imprint any magnetic configuration (as confirmed by theoretical calculations discussed below) and the induced paramagnetic centers were far from each other, precluding the establishment of a long - range magnetic ordering . Thus, the gn0.015 and gn0.033 sample showed dominant diamagnetic behavior, as also witnessed from the isothermal magnetization curves measured at 5 k (see insets in figure 4a, b). Temperature evolution of the mass magnetic susceptibility, mass, of the (a) gn0.015 and (b) gn0.033 sample recorded under an external magnetic field of 1 koe . The insets in panel (a) and (b) show the temperature profile of mass after subtraction of the diamagnetic component and behavior of the 5 k hysteresis loop of the gn0.015 and gn0.033 sample, respectively . (c) temperature evolution of mass for the gn0.051 sample recorded under an external magnetic field of 1 koe with the curie temperature, tc, indicated . The insets show the trend of mass at low temperatures and the temperature profile of the fm contribution, mass, ferro, derived from fitting the measured mass . (d) hysteresis loop of the gn0.051 sample measured at a temperature of 5 k. the insets show the behavior of the hysteresis loop around the origin with the coercivity marked and field - dependent profiles of magnetization for the ferromagnetic, mferro, and paramagnetic, mpara, component derived from fitting the measured isothermal magnetization curve . (e) scheme showing different magnetic fractions in the gn0.051 sample and their respective profiles of mass . Interestingly, for the gn0.051 sample, the temperature profile of mass was drastically different compared to the gn0.015 and gn0.033 sample as it could not be described by the modified curie law . On lowering the temperature, mass showed a saturation tendency followed by an abrupt increase, indicating two contributions, i.e., fm and paramagnetic (see figure 4c e); contrary to the graphene systems with a lower n - doping, the diamagnetic term was found to be negligible here . The presence of both magnetic fractions can be explained by assuming that the n - doped graphene sheets within the sample contained nitrogen in different structural configurations, which have a different impact on the magnetism of graphene, as predicted by the theory discussed below . Moreover, it is known that even in sheets showing fm behavior, isolated paramagnetic centers may evolve owing to the presence of vacancies and defects of topological and edge nature . In order to fit the temperature evolution of mass, the paramagnetic term was fitted using the curie function (i.e., mass = c / t) over the whole temperature interval, whereas a model function involving a combination of the curie weiss law (i.e., mass = c/(t), where is the weiss temperature) at high temperatures and brillouin function within the mean - field approximation at low temperatures was constructed to describe the fm contribution (see inset in figure 4c). The fitting yielded 69 k, angular momentum number j 1.19, and a weight - normalized ratio of ferromagnetic - to - paramagnetic contribution equal to 1.66 . Note that the value of j is not an integral multiple of 0.5 as expected and must be treated as an average over all the n - doped graphene sheets in the gn0.051 sample as the sheets can have different c n configurations / arrangements with different net magnetic moments (see below), as already reported for n - doped and s - doped graphene systems . Furthermore, can be assigned to the curie temperature, tc, marking the transition from the paramagnetic to fm regime on lowering the temperature . Thus, in accordance with the theory, if the concentration of nitrogen exceeds a threshold value (> 5 at .% of n), magnetically active motifs inside the graphene lattice evolve, eventually leading to a magnetically ordered (i.e., fm) state at low temperatures .% of n to the low - temperature fm regime was further confirmed by a series of hysteresis loops measured over the temperature range from 5 to 300 k (see figure 4d and figure s5 in supporting information). At 5 k, the isothermal magnetization curve showed hysteresis with a coercivity of 92 oe (see inset in figure 4d) and saturation magnetization reaching 1.09 emu / g . The derived value of the saturation magnetization is of identical order as reported for s - doped graphenes and vertical graphenes, which are regarded as the magnetically strongest carbon - based systems prepared to date . The nonzero value of the coercivity implies that n - doping imprints magnetic anisotropy on the graphene lattice, establishing an easy axis of magnetization along which the magnetic moments of the generated paramagnetic centers energetically prefer to lie . Following the mathematical procedures of liu et al . And tuek et al . And assuming the j value and weighted ferromagnetic - to - paramagnetic ratio derived from the mass vs t profile, we next attempted to separate the paramagnetic and fm contribution in the 5 k isothermal magnetization curve (see inset in figure 4d). The fitting yielded a saturation magnetization of 0.65 emu / g and 0.37 emu / g for the fm and paramagnetic contributions, respectively . On raising the temperature, the coercivity decreased to zero (see inset in figure s5b in supporting information) and the hysteresis was lost at a temperature of 70 k (see figure s5a in supporting information), indicating a transition from the fm to paramagnetic regime . Thus, the experimental results suggested that when the concentration of nitrogen was increased and it became firmly embedded in the crystal lattice of graphene, the number of induced paramagnetic centers increased, eventually forming magnetically active motifs with conduction electrons providing interaction pathways between them and establishing long - range magnetic ordering upon decreasing the temperature . It was hypothesized that upon further increasing the nitrogen concentration in the graphene lattice, magnetic interactions would be strengthened, as reflected by a shift of tc to higher temperature . Moreover, the presence of more paramagnetic centers would enhance the saturation magnetization, approaching the values observed for s - doped and vertically oriented graphenes, i.e., systems where the magnetic features are imprinted by defects . Here% of n was probably a consequence of the different nitrogen motifs identified in the samples by xps (graphitic, pyridinic, and chemisorbed importantly, we did not identify pyrrolic nitrogen in the samples, which has previously been shown to cause a fall in magnetization values . To decipher the effect of nitrogen in various bonding configurations (as identified by xps, figure 2 and figure s3 in supporting information) on the magnetic properties of graphene, we performed a first - principles study of the structural, electronic, and magnetic properties of n - doped graphenes . The magnetic contributions of individual n - motifs were also addressed in details . We used a rectangular graphene cell containing 96 atoms, which was computationally tractable and enabled the experimentally determined total and relative (among different bonding configurations) content of nitrogen to be followed closely . Specifically, we considered chemisorbed nitrogen (both n and n2) on the top of the graphene sheet and two graphitic and pyridinic nitrogen atoms in the graphene lattice (see figure 5). Top view of graphene doped with graphitic (in para configurations), trimerized pyridinic and chemisorbed (a) n and (b) n2 marked respectively by red, green, and magenta, with positive (negative) spin densities plotted in blue (red) for isosurfaces at 1 10 (panel (a)) and 2.5 10e (panel (b)). (c, d) the corresponding dos plot for configuration shown in panel (a) and panel (b), respectively . The computational data fully supported the experimental results . In particular, the energetically most stable structures, as presented in figure 5a, b, exhibited fm ordering with 1.3 and 0.3 b magnetic moment per supercell for n and n2, respectively, chemisorbed on the graphene surface . The density of state (dos) plots presented in figure 5c, d indicated exchange coupling mediated by the conduction electrons . Moreover, the strong spin - polarized electron features at the fermi level in the dos structures showed a predominant contribution from the graphitic n atoms, which highlights their important role in developing stoner - like magnetism in n - doped graphenes . It should be noted that depending on the position of nitrogen on the graphene surface with respect to the graphitic and pyridinic nitrogen atoms, a large number of possible magnetic configurations were found with a varying effective magnetic moment per cell and exhibiting both fm and antiferromagnetic behavior or even disappearance of magnetic order . This may explain the complexity of the magnetic measurements discussed above for the gn0.051 sample, as the experimental response represented an average over a large number of structures with different net magnetic moments . Thus, to understand the role and contribution of individual nitrogen motifs in triggering magnetism in n - doped graphenes, we carried out an extensive set of additional calculations with a smaller cell containing 48 atoms . The magnetic properties of graphene doped with graphitic nitrogen exhibited a strong dependence on both the nitrogen concentration and configurations .% of graphitic nitrogen (the percentages used result from the size of the cell employed in the calculations) are shown in figure s6 in supporting information . The magnetism of graphene doped with graphitic nitrogen has been attributed to delocalized electrons occupying narrow peaks at the fermi level (ef).% of nitrogen was predicted to be nonmagnetic, in accord with dft calculations by wang et al ., and the narrow electron donor states near ef were absent in the partial density of states (pdos). At 4.2 at .% of nitrogen, the computational screening identified a magnetic structure with a magnetic moment of 0.2 b per supercell in which n atoms substituted c atoms at para positions (see figure 6). The same motif was responsible for triggering ferromagnetism in the larger 96-atom cell, which highlights the importance of the graphitic nitrogen motif in imprinting fm order in n - doped graphene . N - doping generating the fm state gave rise to a strong pz electron peak at the fermi level in the electronic structure according to pdos, similar to recent reports for graphene doped with 4.2 at . The magnetic polarization was confined to a narrow part of the cell, in zigzag directions between the doping atoms, as can be seen in the inset in figure 6 presenting isosurfaces of spin densities . Importantly, formation of zigzag edges by cutting graphene along a certain crystallographic direction has been shown to give rise to peculiar edge localized states near the fermi level responsible for the spontaneous formation of magnetic ordering in graphene nanoribbons . Partial densities of states calculated for graphene doped with nitrogen embedded in the lattice at para positions at a concentration of 4.2 at . The supercell is shown in the inset, where an isosurface of spin - density plotted at 5 10e is also displayed .% of graphitic nitrogen, a greater number of magnetic configurations with the magnetic moment varying between 0.1 and 0.8 b / supercell (see figure 7 and figure s7 in supporting information) were produced . These magnetic structures contained two n atoms in the meta configuration embedded in the graphene lattice . The magnetic behavior of this system could be tuned by changing the position of the third n atom in the host lattice as it stayed on the zigzag paths between the other two n atoms . Finally, because of the similar atomic sizes of nitrogen and carbon atoms, incorporation of nitrogen into the honeycomb network of graphene did not lead to any significant distortion of the host lattice . In - plane atomic displacements caused by nitrogen were below 2% of the c c distance of pristine graphene and the system remained planar . Top view of graphene doped with nitrogen at a concentration of 6.25 at . Positive (negative) spin densities are plotted in blue (red) for isosurfaces at 5 10e: (a) 0.1, (b) 0.4, (c) 0.7, (d) 0.8, (e) 0.4, and (f) 0.6 b per supercell . We also considered the effect of nitrogen atoms substituting carbons in the graphene lattice (employing a 48-atom graphene cell) on pyrrolic bonding sites . A single pyrrolic nitrogen in graphene occupied a site inside the pentagonal ring in the vicinity of single vacancy (sv) or divacancy (dv) defect (see figure s8a, b in supporting information). Substitution by monomeric pyrrolic nitrogen did not result in any long - range magnetic order . C bond with an n atom led to opening of the 5-membered ring and s = 1/2 paramagnetism due to the carbon dangling bond . Such a bonding configuration transformed the initial pyrrolic nitrogen into pyridinic nitrogen (see figure s8c, d in supporting information). We also considered the effect of adding pyrrolic nitrogen inside the octagonal ring of the dv defect (figure s8e, f in supporting information), as proposed in the work of li et al . The present calculations indicated that the nitrogen atom moved to the neighboring 5-membered ring, in accord with the study by lin et al ., forming a 6-membered ring upon relaxation in which nitrogen (transformed into pyridinic) was nonmagnetic . Next, we considered the role of pyridinic nitrogen, which was also identified as an important motif in the experimental samples . Besides monomeric pyridinic nitrogen (s = 0 or s = 1/2 due to carbon dangling bonds), we also considered dimerized (with s = 1/2 magnetic moment due to carbon dangling bond) and trimerized pyridinic nitrogen (with a magnetic moment of 0.3 b per supercell). Note that the calculated ground state arrangements of multiple pyridinic nitrogen in the graphene lattice were in agreement with annular dark - field imaging reported in the work by lin et al . However, according to dos of the trimerized pyridinic nitrogen (see figure s9 in supporting information), the electronic structure indicated that indirect exchange mediated by the conduction electrons was strongly compromised compared to the exchange coupling developed for graphene doped with graphitic nitrogen (compare figure 6 and figure s9 in supporting information). This is in line with the suppressed magnetic moment of trimerized pyridinic nitrogen in comparison to that of trimerized graphitic nitrogen (see the structure presented in figure 7f and dos plot in figure s7f in supporting information). It is also important to mention that half - metallic properties were recently predicted for the graphene - based c4n3 polymer, i.e., a 2d radical polymer containing many trimerized pyridinic nitrogens . In contrast, the dos plot presented in figure s9 in supporting information showed finite (nonzero) density of states above ef in both spin - up and spin - down channel, which is most likely due to a significantly lower concentration of trimerized pyridinic nitrogen in the present study . Finally, we investigated whether magnetism in graphene can also be induced by nitrogen adatoms / molecule, which were identified in the xps patterns and tga / ega measurements . Thus, structures resulting from both nitrogen additions on a pristine graphene layer and simultaneous single nitrogen atom addition and graphitic substitution were analyzed (see figure s10 and figure s11 in supporting information). A single n - adatom carried a magnetic moment of 0.5 b, which can be understood based on an electron counting argument: two valence electrons formed covalent bonds with neighboring c atoms, two formed a lone - pair, and the fifth valence electron gave rise to the magnetic moment . Another nitrogen atom contributed 0.5 b, but no magnetic moments were induced on the c atoms . By placing another nitrogen atom in a close proximity to the preadsorbed adatom, an n2 dimer spontaneously formed, which was adsorbed over the surface and had zero magnetic moment .% of graphitic nitrogen (see figure s11 in supporting information) also did not induce a magnetic response in the system . To conclude, the computational study allowed elucidation of the synergistic effect of nitrogen atoms in various bonding configurations, as evidenced from high - resolution xps data . To follow closely the total and relative content of nitrogen in the experimentally prepared samples, we considered graphitic nitrogen in the para configuration, which turned out to promote formation of the motif most important for imprinting magnetism in graphene, followed by trimerized pyridinic nitrogen and chemisorbed n adatoms . In the ground state, the structure resulting from the combined effects of all these species is strongly fm . However, it should be noted that coupling between pyridinic nitrogens is much less effective in maintaining the fm structure, and chemisorbed nitrogen adatoms can only generate paramagnetism . Finally, pyrrolic nitrogen has no effect on magnetism in graphene, in line with the work by ito et al ., who reported a decrease in the magnetization values with increasing concentration of pyrrolic nitrogen in the graphene lattice . In summary, on the basis of electronic - structure calculations and magnetization measurements, we have provided new insights into the role of nitrogen as a highly electronegative n - type dopant for imprinting the magnetic properties to graphene . The magnetic features of n - doped graphene depend on both the nitrogen concentration and the configuration in the host lattice, with a complex interplay between graphitic, pyridinic, and chemisorbed nitrogen . Among these structural motifs,% of nitrogen, graphene behaves dominantly as a diamagnet; paramagnetic centers are induced upon doping; however, they do not produce magnetically active motifs . If the doping concentration is increased above the threshold doping value, magnetic interactions mediated by the conduction electron system emerge between the substitution - generated paramagnetic centers .% shows a transition to an fm state at the curie temperature of 69 k and saturation magnetization reaching 1.09 emu / g . Such a high value of the saturation magnetization ranks n - doped graphenes among the magnetically strongest graphene - based systems developed so far for which the magnetic properties are imprinted by defects . As n - doping is also expected to maintain or even improve the electric (i.e., conduction) features of graphene, the present work opens possibilities for further optimization of n - doped graphenes (e.g., exclusive presence of graphitic nitrogen) to produce new kinds of spintronic materials.
Sphingosine 1-phosphate (s1p) is a biologically active lysophospholipid which is produced by the metabolism of sphingolipid catalyzed by sphingosine kinase . S1p is involved in various cell activities including cell differentiation, apoptosis, proliferation, migration, and others . The roles of s1p have recently drawn attention as s1p has been discovered to be a critical regulator of bone metabolism by many studies . First, s1p is released from osteoclasts and facilitates the proliferation, migration, and survival of osteoblasts by working as a coupling factor between osteoblasts and osteoclasts. [2 - 4] second, s1p mediates bone destruction by inducing the formation of osteoclasts . Third, s1p is more abundant in circulating blood than bone marrow cavity. [6 - 8] when the difference of s1p levels between those two increases, osteoclast precursors are limitedly present in bone marrow cavity, promoting bone resorption. [9 - 12] however, the role of s1p in humans and the relative importance of three mechanisms mentioned in the above have not been clarified yet . In relation to this, the authors of this study have recently reported that bone resorption is promoted and bone mineral density (bmd) decreases as plasma s1p levels become higher in the human body . These previous findings imply that s1p is not only important to humans but also, involved in two mechanisms facilitating bone resorption by directly promoting bone resorption and maintaining constant concentration difference between circulation system and bone marrow cavity . In addition, the findings suggest that one of the bone resorption mechanisms is more important than the other and the bone formation - related mechanism . This investigation is a preliminary study investigating if which mechanism is more significantly involved between two bone resorption mechanisms . The study aims to identify the differences in s1p levels by collecting bone marrow in patients who underwent surgeries due to osteoporotic fractures and causes with other than osteoporotic fractures . This study involved 16 patients who underwent hip surgery in the department of orthopedic surgery, asan medical center from july 2012 to september 2012 . We excluded patients who took osteoporosis medications affecting bone metabolism for six months, and patients with diseases that develop secondary osteoporosis including hyperthyroidism and others . Osteoporotic hip fracture excluded high - impact fractures induced by car accident and others, and four subjects fell under this category (table 1). The study was carried out after gaining the institutional review board (irb) approval of asan medical center and written consent form of all subjects . The study investigated sex, age, body weight, and height of all subjects and identified their diagnosis at the time of operation . Bone marrow collected during surgery was stored in -70 deep freezer by dividing into 200 l . Bone marrow samples were centrifuged at 3,000 rpm for 5 minutes at 4 to separate bone marrow fluids for the measurement of s1p levels (mol / l). Centrifuged bone marrow fluids were kept at -70 before the measurement . S1p competitive enzyme - linked immunosorbent assay (elisa) kit (echelon biosciences inc ., salt lake city, ut, usa) was used for the measurement of s1p levels . Mol / l, and inter- and intra - measurement coefficients of variation were 6.4% and 6.1%, respectively . Bone marrow s1p level of each patient was measured twice and the average value was used for analysis . Serum calcium (mmol / l) and phosphorus levels (mmol / l) were measured by applying cresolphthalein comlexone method and phosphomolybdate ultraviolet (uv) method, respectively, using toshiba 200fr autoanalyzer (toshiba medical systems co., ltd ., glomerular filtration rate (gfr) (ml / min/1.73 m) was obtained using the cockroft - gault formula . Bmd (g / cm) was measured in lumbar spine and proximal femur (femoral neck, total hip) using dual energy x - ray absorptiometry (lunar prodigy advance, ge lunar, medison, wi, usa). The differences of mean values between two groups were analyzed using an independent samples t - test, and the adjustment of confounding factors was performed with the analysis of covariance . Partial correlation analysis was performed to verify the association bewteen bone marrow s1p levels and bmds, and three analysis models were established for the adjustment of confounding factors . Age and sex were included in model 1, body weight and height were added in model 2, and serum calcium, phosphorus levels and gfr were added in model 3 . P - values of less than 0.05 were considered to be statistically significant in all statistical analysis . This study involved 16 patients who underwent hip surgery in the department of orthopedic surgery, asan medical center from july 2012 to september 2012 . We excluded patients who took osteoporosis medications affecting bone metabolism for six months, and patients with diseases that develop secondary osteoporosis including hyperthyroidism and others . Osteoporotic hip fracture excluded high - impact fractures induced by car accident and others, and four subjects fell under this category (table 1). The study was carried out after gaining the institutional review board (irb) approval of asan medical center and written consent form of all subjects . The study investigated sex, age, body weight, and height of all subjects and identified their diagnosis at the time of operation . Bone marrow collected during surgery was stored in -70 deep freezer by dividing into 200 l . Bone marrow samples were centrifuged at 3,000 rpm for 5 minutes at 4 to separate bone marrow fluids for the measurement of s1p levels (mol / l). Centrifuged bone marrow fluids were kept at -70 before the measurement . S1p competitive enzyme - linked immunosorbent assay (elisa) kit (echelon biosciences inc ., salt lake city, ut, usa) was used for the measurement of s1p levels . Mol / l, and inter- and intra - measurement coefficients of variation were 6.4% and 6.1%, respectively . Bone marrow s1p level of each patient was measured twice and the average value was used for analysis . Serum calcium (mmol / l) and phosphorus levels (mmol / l) were measured by applying cresolphthalein comlexone method and phosphomolybdate ultraviolet (uv) method, respectively, using toshiba 200fr autoanalyzer (toshiba medical systems co., ltd ., glomerular filtration rate (gfr) (ml / min/1.73 m) was obtained using the cockroft - gault formula . Bmd (g / cm) was measured in lumbar spine and proximal femur (femoral neck, total hip) using dual energy x - ray absorptiometry (lunar prodigy advance, ge lunar, medison, wi, usa). The differences of mean values between two groups were analyzed using an independent samples t - test, and the adjustment of confounding factors was performed with the analysis of covariance . Partial correlation analysis was performed to verify the association bewteen bone marrow s1p levels and bmds, and three analysis models were established for the adjustment of confounding factors . Age and sex were included in model 1, body weight and height were added in model 2, and serum calcium, phosphorus levels and gfr were added in model 3 . P - values of less than 0.05 were considered to be statistically significant in all statistical analysis . Three patients (75%) were females among 4 patients with osteoporotic hip fracture (table 1). Among 12 patients who underwent operation due to causes other than osteoporotic hip fracture, 7 patients were osteoarthritis, 4 patients were avascular necrosis (avn) of the femoral head, and a patient was hip dislocation . There were a male (25.0%) and three female (75.0%) patients with osteoporotic hip fractures . Mean age was 64.99.8 years, showing younger mean age (p=0.607), and bmi was 25.13.9 kg / m, showing a higher tendency (p=0.050). The average bone marrow s1p level of all subjects was found to be 3.401.10 mol / l (range: 2.08 - 5.74 we compared bone marrow s1p levels of two groups according to the presence of osteoporotic hip fractures (fig . Bone marrow s1p levels were 2.480.38 mol / l in patient group with osteoporotic hip fracture, and 3.701.09 mol / l in control group . Bone marrow s1p level was significantly low in patients with osteoporotic hip fracture (p=0.047) (fig . Bone marrow s1p levels were 2.311.94 mol / l in patient group with osteoporotic hip fracture and 3.761.10 mol / l in control group, showing significance (p=0.025) (fig . When we analyzed the correlation between bone marrow s1p level and bmd, statistically significant correlation was not observed after the adjustment of several confounding factors (=0.026 - 0.482, p=0.171 - 0.944) (table 2). This study identified that the bone marrow s1p levels were significantly lower in patients with osteoporotic hip fractures than in those without . No studies have been performed to verify the association between the presence of osteoporosis and osteoporotic fracture by clinically measuring s1p levels in the bone marrow of humans . The investigation is the first study to observe the positive effects on bone metabolism of high s1p levels in bone marrow, contradicting previous in vivo studies that suggested that s1p directly increases osteoclast differentiation and bone resorption . In a recent study, the authors have verified that bmds decrease and bone resorption markers increase as plasma s1p levels become higher in all patients including men, and pre- and postmenopausal women . In contrast, this investigation found the reduction in the prevalence of osteoporotic fracture as bone marrow s1p levels increase . Thus, the risk of fracture may increase as s1p levels rise in peripheral blood . However, the risk of fracture may be reduced when bone marrow s1p level is higher . To sum up the above study results, s1p level is not directly involved in bone cells, instead bone resorption is accelerated as the concentration difference of s1p between peripheral blood and bone marrow cavity increases, supporting the hypothesis . High plasma s1p concentration is maintained as s1p is consistently released by vascular endothelial cells, red blood cells (rbcs), platelet, and others . On the other hand, concentration difference between plasma and tissue is formed by maintaining low s1p levels with constant breakdown catalyzed by intracellular s1p lyase or s1p phosphatase in each tissue including bone marrow . The s1p receptor type 2 (s1pr2) expression of osteoclast precursors is dominant in peripheral blood with high s1p level, and it promotes the migration of osteoclast precursors to bone marrow by triggering the signaling pathways of g12/13-mediated rho activation . Once osteoclast precursors migrate into bone marrow, they are differentiated to osteoclasts . Therefore, the migration of osteoclast precursors to bone surface is accelerated as the difference of s1p level between peripheral blood and bone marrow is higher, leading to higher risk of fracture due to increased bone resorption . This study failed to demonstrate significant association between bone marrow s1p levels and bmd values . However, the study involved a relatively small number of subjects, measuring bmds only in 14 patients . Taking this fact into consideration, the results could not be interpreted that s1p is not taking a crucial role in bone metabolism . There is also limitation in generalizing the study results obtained from small sample size of 16 patients . Furthermore, the concentration difference between peripheral blood and bone marrow is more critical factor involved in osteoclast migration rather than s1p present in bone marrow . However, the study was unable to measure s1p levels in the blood of same patient . Therefore, this investigation is considered as a preliminary study suggesting the conduction of clinical trials in the future . This study verified that bone marrow s1p level was lower in patient group with osteoporotic hip fractures compared with control group . These suggest that the difference of s1p levels between peripheral blood and bone marrow is critical on bone metabolism.
Gene duplication plays a key role in evolution by providing dna templates for evolutionary innovation, while preventing interference with the cellular function of the original genes (13). Immediately after gene duplication, the resulting paralog pairs are usually identical and therefore functionally redundant . Unless duplication confers a selective advantage, either via gene dosage effects or via mutational acquisition of modified or new functions, duplicated genes will eventually be pseudogenized and/or lost from the genome (2, 3). Gene duplication played a key role in the evolutionary history of the yeast saccharomyces cerevisiae, an intensively investigated model in eukaryotic evolutionary biology . A whole - genome duplication event (wgd) in an ancestor of s. cerevisiae, ca . Despite the long time interval that separates current s. cerevisiae strains from the wgd, many surviving paralog pairs still exhibit a substantial degree of functional redundancy, as indicated by neutral or weak phenotypes of single - paralog deletion mutants (69). However, the selective pressures that caused the long - term retention of these functionally redundant paralogs remain elusive (1, 10). The embden - meyerhof - parnas (emp) pathway, the main route for oxidation of glucose to pyruvate in all eukaryotes and in many other organisms, is among the most slowly evolving metabolic pathways (11, 12). In all taxa, paralog families are found for the structural genes that encode the emp enzymes (1214). Under conditions of oxygen limitation and/or sugar excess, s. cerevisiae couples the emp pathway to the fermentative production of ethanol via pyruvate decarboxylase and nad - dependent alcohol dehydrogenase (15, 16). In this article, we use the term glycolysis to indicate the set of 12 enzyme - catalyzed reactions in yeast that convert intracellular glucose to ethanol . In s. cerevisiae, no fewer than 8 of the 12 enzyme reactions in glycolysis are represented by multiple paralogous genes (fig . 1). This incidence represents a significant overrepresentation (p = 1.9 10, based on hypergeometric distribution analysis) relative to the ca . The wgd event and resulting duplication of genes involved in central metabolism have been implicated in the appearance of several key physiological characteristics of s. cerevisiae . In particular, the duplication of glycolytic genes has been proposed to have contributed to the strong tendency of s. cerevisiae to produce ethanol under aerobic conditions (crabtree effect) and its high glycolytic capacity (1820). However, the impact of reducing the number of glycolytic paralogs on these and other physiological characteristics of s. cerevisiae has not been systematically explored . In all paralogous gene sets in yeast glycolysis, with the notable exception of the phosphofructokinase gene, gene expression and gene deletion studies support the definition of a single major paralog and one to four minor paralogs (fig . All paralogs have retained their original catalytic function, although their context - dependent expression profiles differ (fig . Deletion of minor paralogs for individual glycolytic enzymes has minor effects on enzyme activities in cell extracts and on the specific growth rate under standard laboratory conditions (usually shake flask cultivation on yeast extract, peptone, and glucose) (2125) (fig . Several hypotheses have been forwarded to explain the neutral effect of paralog deletion (6, 2631). These include experimental limitations, such as the poor sensitivity of fitness screens (32) and the narrow range of cultivation condition tested (generally complex medium) (33). Additionally, analysis of deletion mutants in which paralogs that encode a single glycolytic enzyme are inactivated cannot reveal synergistic effects of the minor paralogs of different glycolytic enzymes . Such synergistic effects might, for example, arise from the well - documented phenomenon of distribution of metabolic control over multiple enzymes in metabolic pathways (34, 35) or from other regulatory or catalytic interactions . The following eight enzymes in yeast glycolysis are encoded by parologous genes: hexokinase / glucokinase (hxk), phosphofructokinase (pfk), glyceraldehyde-3-phosphate dehydrogenase (gapdh), phosphoglycerate mutase (gpm), enolase (eno), pyruvate kinase (pyk), pyruvate decarboxylase (pdc), and alcohol dehydrogenase (adh). Percentages of gene and protein similarity (gesy and prsy, respectively) between paralogs, the type of duplication event (duev), whole - genome duplication (wgd) or other small scale duplication (ssd), and the proposed fate (pseudogenization [p], subfunctionalization [s], or neofunctionalization [n]) of the different paralogs are indicated in the tables in each panel . Separation between wgd and small - scale duplications pre - wgd (ssd ) and post - wgd (ssd) was based on the information in the yeast gene order browser (ygob; http://ygob.ucd.ie/) (119). Scatter plots show the comparisons between expression levels of the different glycolytic paralogs in s. cerevisiae measured under 170 different conditions (75) (see table s6 in the supplemental material). Except for pfk, where both paralogs pfk1 and pfk2 were considered paralogs with equivalent contributions, the expression levels of the major glycolytic paralogs (labeled in red) are indicated by a red line . Bar graphs display in vitro enzyme activities of glycolytic enzymes in mutants carrying individual and multiple glycolytic gene knockouts . Values are presented as percentages of in vitro enzyme activities measured in reference strains (rs [denoted by an orange dotted line]) as reported in the literature (2125). Insight into the importance, under laboratory conditions, of the minor glycolytic paralogs is essential for understanding an apparent genetic redundancy in a key ubiquitous metabolic pathway in an important model organism and industrial platform . In addition, analysis of the extent of genetic redundancy in central metabolism is highly relevant for the complete redesign and construction of entirely synthetic yeast genomes, as pursued in the synthetic yeast 2.0 initiative (36). Moreover, if complexity in yeast glycolysis can be significantly reduced by elimination of redundant isoenzymes, this could eliminate uncertainties and thereby facilitate the formulation and validation of mathematical models that describe the kinetics of this key metabolic pathway (37, 38). The goals of the present study are to experimentally explore genetic redundancy in yeast glycolysis by cumulative deletion of minor paralogs and to provide a new experimental platform for fundamental yeast research by constructing a yeast strain with a functional minimal glycolysis (mg). To this end, we deleted 13 minor paralogs, leaving only the 14 major paralogs for the s. cerevisiae glycolytic pathway . The cumulative impact of deletion of all minor paralogs was investigated by two complementary approaches . A first, quantitative analysis focused on the impact on glycolytic flux under a number of controlled cultivation conditions that in wild - type strains result in different glycolytic fluxes . These quantitative growth studies were combined with transcriptome, enzyme activity, and intracellular metabolite assays to capture potential small phenotypic effects . A second, semiquantitative characterization explored the phenotype of the minimal glycolysis (mg) strain under a wide array of experimental conditions to identify potential context - dependent phenotypes . Plasmid propagation and isolation were performed with chemically competent escherichia coli dh5 (z - competent transformation kit; zymo research, orange, ca) cultivated in lysogeny broth (lb) medium (39, 40) supplemented with 100 mg liter ampicillin (lbamp) when required . All yeast strains are derived from the cen.pk family (4143) and are listed in table s3 in the supplemental material . All strains were stored at 80c in 1-ml aliquots of 30% glycerol and the appropriate medium . Cen.pk102-12a was selected as the parental strain for the minimal glycolysis (mg) strain . The order of gene deletions that led to the final mg strain imx372 was glk1, hxk1, tdh1, tdh2, gpm2, gpm3, eno1, pyk2, pdc5, pdc6, adh2, adh5, and adh4 . The genes glk1, hxk1, tdh1, and tdh2 were deleted using the auxotrophic and dominant markers sphis5 (44, 45), klleu2 (45, 46), kanmx (47, 48), and hphnt1 (49, 50), respectively . Gpm2 to adh4 deletions were performed using a strategy of selection and counterselection with the klura3/5-fluoroorotic acid (5-foa) system (51) for the recovery of the marker module . The dominant marker modules kanmx and hphnt1 were removed using deletion cassettes containing klura3 and amdsym, respectively . These markers were sequentially removed as reported previously (54). To obtain a prototrophic strain, a cassette containing the marker module klura3 was integrated in the tdh1 locus; this generated the mg strain . All integrative cassettes were constructed using phusion hot start ii high - fidelity polymerase (thermo scientific, landsmeer, the netherlands) following the manufacturer's recommendations and with the primer pairs and plasmids listed in tables s4 and s5 in the supplemental material, respectively, as the templates . Correct integrations, deletions, and marker excision were confirmed by pcr using dreamtaq polymerase (thermo scientific) and following the manufacturer's recommendations . Genomic dna that served as the template for pcr was obtained by extraction with 0.05 n naoh directly from single colonies or by purification using the yeastar genomic dna kit (zymo research, orange, ca) following the manufacturer's recommendations . All pcr products were loaded on gels containing 1% (wt / vol) agarose (thermo scientific) and 1 tris - acetate - edta (tae) buffer (thermo scientific). Integrative cassettes were gel purified using the zymoclean gel dna recovery kit (zymo research). Yeast strain transformations were performed with the lithium acetate protocol as previously described (55). Plasmids were isolated from e. coli with the genelute plasmid miniprep kit (sigma - aldrich, st . Complex and nonselective media for growth rate determination and propagation contained 10 g liter yeast extract, 20 g liter peptone, and 20 g liter glucose (ypd). When selection was required, ypd was supplemented with 200 mg liter g418 (ypd+g418) or 200 mg liter hygromycin (ypd+hyg). Synthetic medium (sm) containing 3 g liter kh2po4, 0.5 g liter mgso47h2o, 5 g liter (nh4)2so4, 1 ml liter of a trace element solution, and 1 ml liter of a vitamin solution as previously described (56). Sm was supplemented with 20 g liter glucose (smg) for propagation, growth rate determination, and batch cultures . When auxotrophic strains were cultivated, smg was supplemented with 150 mg liter uracil, 125 mg liter histidine, and/or 500 mg liter leucine, according to strain auxotrophy (57). Selection of strains lacking the klura3 marker was done in smg supplemented with 150 mg liter uracil and 1 g liter 5-fluoroorotic acid (smg5-foa). Unless otherwise stated, liquid cultures were performed in 500-ml shake flasks with a working volume of 100 ml and incubated at 30c and 250 rpm, the targeted starting optical density at 660 nm (od660), measured using a libra s11 spectrophotometer (biocrom, cambridge, united kingdom), being 0.5 . Unless otherwise stated, cultures on solid media were incubated for 3 days at 30c . Growth of the mg strain and of the prototrophic control strain cen.pk113-7d was performed on agar plates containing ypd or sm plus 2% glucose (smg), sm plus 2% galactose (smgal), sm plus 2% maltose (smmal), sm plus 2% sucrose (smsuc), sm plus 3% (vol / vol) ethanol (smetoh), sm plus 6% glycerol (smglyc), sm plus 5% glucose (smhg), sm plus 0.5% glucose (smlg), smg plus 2.5, 5, or 10 mm licl (smg - lit), smg plus 200 or 500 mm nacl (smgsa), smg plus 50, 100, or 200 m cdcl2 (smgcad), smg plus 1, 1.5, or 2 m sorbitol (smgsor), smg at ph 4, 6, or 7.5 (smgph), or smg plus 1 mm h2o2 (smgox). Spot plates were inoculated with 10 to 10 cells obtained by serial dilutions of liquid cultures grown to the exponential phase on smg . All plates were incubated at 30c for 3 days, except for plates containing smglyc, which were incubated for 6 days . Growth rate determination was performed by od660 measurement of cultures grown in 500-ml shake flasks containing 100 ml of ypd, smg, or smetoh . Cultures for growth rate measurements were inoculated with precultures grown until the late exponential phase under identical conditions (i.e., the same medium and temperature). Glucose / ethanol / glucose switches were started by inoculation of smg shake flasks with a preculture grown overnight on the same medium . At the mid - exponential phase (od660 of ca . 4) samples were taken, washed twice with sterile demineralized water, and used to inoculate shake flasks containing 100 ml of smetoh . When reaching an od660 of 3, a sample from the culture was washed twice with sterile demineralized water and used to inoculate a 500-ml shake flask containing 100 ml smg . Growth was then monitored until the late exponential phase . To evaluate tolerance of mg to oxidative stress, cells grown to the exponential phase in shake flasks containing smg were transferred to a shake flask containing smg supplemented with 4.5 mm h2o2 . Samples were then taken every 20 min for 1 h, diluted, and plated on smg plates to reach approximately 100 cells per plate . Aerobic and anaerobic batch cultures were performed in 2-liter laboratory fermenters (applikon, schiedam, the netherlands) with a 1-liter working volume . Antifoam emulsion c (sigma) was added to a concentration of 0.2 g liter from a 20% (vol / vol) solution autoclaved separately (121c). Prior to inoculation, glucose was added to a final concentration of 20 g liter from a sterile (110c) 50% glucose solution and 1 ml of a vitamin solution (56). Anaerobic cultures were supplemented with 0.01 g liter ergosterol and 0.42 g liter tween 80 dissolved in ethanol as previously described (56). Compressed air or gaseous nitrogen (quality, 4.5;> 99.995% vol n2, <10 volumes per million [vpm] o2 pollution) (linde gas, schiedam, the netherlands) for aerobic and anaerobic cultures, respectively, was sparged to the bioreactor at a rate of 500 ml min via an ion science saga digital flow meter (cambridge, united kingdom). Precultures were started by inoculation of smg shake flasks with 1 stock vial of the appropriate strain . 16 h of incubation, these cultures were used to inoculate new smg shake flasks . The starting od660 was tightly controlled to reach an od660 of 1 after 8 h of incubation . These exponentially growing cultures were washed twice with demineralized water (spinning at 5,000 rpm for 4 min) and resuspended in 100 ml demineralized water . This was the cell suspension that was used to inoculate fermenters with a starting od660 of 0.1 . The concentration of extracellular metabolite in culture supernatants was measured by high - performance liquid chromatography (hplc) analysis using a aminex hpx-87h ion - exchange column operated at 60c with 5 mm h2so4 as the mobile phase at a flow rate of 0.6 ml min . Agarose plugs for the different strains were prepared using the contour - clamped homogenous electric field (chef) yeast genomic dna plugs kit (bio - rad, richmond, ca), following the manufacturer's recommendations, and used for chef electrophoresis . Mg and cen.pk113-7d vacuoles were stained with the red fluorescent dye fm4 - 64 (excitation / emission, 515/640 nm) (thermo fisher scientific) following the manufacturer's recommendations . Yeast cells and vacuoles were visualized with an imager - z1 microscope equipped with an axiocam mr camera, an ec plan - neofluar 100/1.3 oil ph3 m27 objective, and the filter set bp 535/25, ft 580, and lp 590 (carl - zeiss, oberkochen, germany). Samples equivalent to 62.5 mg of dry weight biomass taken at the mid - exponential phase (ca . 10 h after inoculation) of aerobic batch cultures were used to obtain cell extracts as previously described (58). Measurement of the activity of glycolytic enzymes was carried out as previously described (59), except for phosphofructokinase, the activity of which was determined as described in reference 60 . Enzyme activities are expressed as micromoles of substrate per minute per milligram of protein or units per milligram of protein . Protein concentrations in cell extracts were determined according to reference 61 with bovine serum albumin as a standard . Samples (1.2 ml each) were taken from aerobic batch cultures using a rapid sampling setup (62) and placed directly into vials containing 5 ml 80% methanol precooled at 40c . The samples were washed with precooled 80% methanol, and the extraction was performed with boiling ethanol as previously described (63). C - labeled cell extract was used as an internal standard for metabolite quantification (64). Intracellular glucose (gluc), glucose-6-phosphate (gluc-6-p), fructose-6-phosphate (fruc-6-p), dihydroxyacetone phosphate (dhap), glyceraldehyde-3-phosphate (gap), 3-phosphoglycerate (3pg), 2-phosphoglycerate (2pg), and pyruvate (pyr) were measured by gas chromatography - mass spectrometry (gc - ms) according to reference 65 . Intracellular fructose-1,6-bisphosphate (fruc-1,6-bp) and phosphoenolpyruvate (pep) were measured by liquid chromatography - mass spectrometry (lc - ms) according to reference 66 . Samples for transcriptome sequencing (rna - seq) were obtained from aerobic batch cultures at the mid - exponential phase of growth on glucose (ca . Sampling, rapid quenching in liquid nitrogen, and rna extraction were performed as previously described (68). Sequencing was performed with the illumina hiseq 2500 and carried out by baseclear (leiden, the netherlands). A data set of 51-bp single reads of at least 1 the data were aligned to the reference using the burrow - wheeler alignment tool bwa (69, 70). Gene expression levels were estimated using fragments per kilobase per million (fpkm) values by the cufflinks software (71). To identify differential gene expression between strains cen.pk113-7d and imx372, rna - seq data comparison was performed and statistically assessed using cuffdiff (71). High - quality genomic dna of the strains cen.pk102-12a and imx372 (mg) was obtained using the qiagen 100/g kit (qiagen, hilden, germany) following the manufacturer's recommendations . Libraries of 300-bp inserts were constructed and paired end sequenced (100-bp reads) using an illlumina hiseq 2500 sequencer (baseclear bv, leiden, the netherlands). A minimum data quantity of 950 mb was generated, representing a minimum 80-fold coverage . The sequence reads were mapped onto cen.pk113-7d genome (42) using the burrows - wheeler alignment tool bwa and further processed using samtools (69, 70). Default settings were used, except that the maximum read depth was set to 400 (d400). To minimize false - positive mutation calls, custom perl scripts were used for further mutation filtering as follows: (i) mutation calls containing ambiguous bases in mapping consensus were filtered out, (ii) only the single - nucleotide variations with a quality of at least 20 were kept (with variant quality defined as the phred - scaled probability that the mutation call is incorrect [72, 73]), (iii) mutations with a depth of coverage below 10 were discarded, and (iv) the mutations found in cen.pk102-12a were subtracted from the list sequence . Eventually, the single - nucleotide variations were physically positioned and functionally annotated according to the cen.pk113-7d sequence annotation . The rna - seq data generated in this study have been submitted to the genome expression omnibus database and assigned accession no . The sequence data generated in this study are searchable at ncbi entrez (http://www.ncbi.nlm.nih.gov/) under bioproject prjna269221 . To enable construction of a minimal glycolysis (mg) yeast strain, the first goal was to identify the major paralogs that should be retained in the final strain design . This assessment was based on information from the literature on phenotypes of relevant deletion mutants and on nonglycolytic roles (moonlighting functions) (74) of glycolytic isoenzymes . Furthermore, a compendium of s. cerevisiae transcriptome data obtained under a wide range of controlled cultivation conditions (75) was used to compare expression profiles of glycolytic paralogs . Major paralogs were elected based on the following, nonexclusive criteria (fig . 1; see fig . S1 in the supplemental material): (i) the highest transcript level over a range of growth conditions, (ii) the most extensive loss of enzyme activity in cell extracts upon deletion, (iii) moonlighting functions with a strong impact on specific growth rate or robustness (hxk2, eno1, and eno2) (76, 77), and (iv) the strongest decrease in specific growth rate upon deletion . Based on these criteria, 11 of the 27 glycolytic genes in s. cerevisiae were assessed to be major paralogs: hxk2, pgi, fba1, tpi1, tdh3, gpm1, eno2, pgi1, pyk1 (cdc19), pdc1, and adh1, while 13 minor paralogs (i.e., hxk1, glk1, tdh1, tdh2, gpm2, gpm3, eno1, pyk2, pdc5, pdc6, adh2, adh4, and adh5) were selected for gene deletion . In s. cerevisiae, the pkf1 and pfk2 paralogs have evolved into subunits of a hetero - octameric phosphofructokinase (78). Since deletion of either gene substantially decreases fitness (7981) (see fig . S1 in the supplemental material), both were retained in the minimal glycolysis design . Adh3 encodes a mitochondrial alcohol dehydrogenase involved in an anaerobic redox shuttle across the mitochondrial inner membrane (82). To prevent reduced growth rates under anaerobic conditions, sequential deletion of the 13 minor paralogs in a haploid s. cerevisiae strain belonging to the cen.pk family (4143) yielded the prototrophic strain imx372, which we will refer to as the mg resequencing of the genome of the mg strain confirmed the correct deletion of all 13 minor glycolytic genes . Although transformation of s. cerevisiae can be mutagenic (83, 84), only 20 single - nucleotide differences were detected in the mg strain relative to its ancestor strain, cen.pk102-12a . Eleven of these differences occurred within open reading frames (orfs), of which 10 resulted in amino acid changes (see table s1 in the supplemental material). Whole - genome sequencing and karyotyping indicated the duplication of two short sections of chromosome iii (16.5 kbp, from ycr019w to ycr027c) and chromosome v (19.1 kbp, from yer093c - a to yer104w) (see fig . These regions do not carry genes involved in central carbon metabolism, and no interchromosomal rearrangements were observed . To explore the physiological impact of the simultaneous deletion of all 13 minor glycolytic paralogs, we quantitatively compared specific growth rates, product formation, and gene expression in the mg strain and in a congenic, prototrophic reference strain with a full complement of glycolytic genes . In these studies, a synthetic, chemically defined medium was used, since complex media do not enable cells to express their full genetic potential (33) and complicate quantitative physiological analysis . In aerobic, glucose - grown bioreactor batch cultures, specific rates of growth, substrate consumption, and product formation were not significantly affected by deletion of all 13 minor glycolytic paralogs (fig . The only exception to this observation concerned acetate production, which was slightly higher in the mg strain . Also after the diauxic shift, where the glycolytic flux changes direction as the aerobic yeast cultures consumed the ethanol and acetate produced during the initial growth phase on glucose, the biomass formation and substrate consumption rates of the two strains were virtually identical . In anaerobic yeast cultures, the absence of oxidative phosphorylation makes glycolysis the only pathway for energy conservation . Also under these more demanding conditions, the specific growth rate and metabolic fluxes of the mg strain did not differ significantly from those of the congenic reference strain (fig . Biomass production and extracellular metabolite profiles from aerobic and anaerobic batch cultures in bioreactors of the minimal glycolysis (mg) strain and a congenic reference strain (indicated by c). Shown are biomass and extracellular metabolite profiles from aerobic (a) and anaerobic (b) controlled batch cultures of the minimal glycolysis strain (mg) (open circles) and the prototrophic reference strain cen.pk113-7d (closed circles). The data shown in the graphs are the average and average deviation of the mean from two independent cultures for each strain . The specific growth rate ([per hour]) and biomass - specific rates of glucose consumption (qs), ethanol production (qetoh), glycerol production (qglyc), and acetate production (qace) (all expressed in millimoles per gram dry weight per hour) represent the average and standard deviation of data from four independent cultures for each strain . , statistically significant (by two - tailed t test assuming the same variance in the populations) differences between the two tested strains (p = 0.03 and p = 0.02 for and qace, respectively, in aerobic cultures). To further compare growth of the mg strain in aerobic bioreactor batch cultures, its glycolytic enzyme activities in cell extracts, intracellular metabolite concentrations, and transcriptome remarkably, no significant differences were observed in the transcript levels of any of the major glycolytic paralogs or in vitro glycolytic enzyme activities (fig . 3). With the exception of slightly higher intracellular concentrations of acetaldehyde, concentrations of glycolytic intermediates in the mg strain did not significantly differ from those in the reference strain . To explore potential impacts of the deletion of 13 minor glycolytic paralogs outside glycolysis, genome - wide transcript levels of the mg strain and the reference strain were compared . As few as 17 genes showed a significantly different transcript level (see table s2 in the supplemental material), 12 of which were located on the duplicated regions on chromosomes iii and v. an in - depth, comprehensive analysis of the mg strain in glucose - grown bioreactor batch cultures therefore failed to identify substantial impacts of the minor glycolytic paralogs on fluxes, intracellular metabolite concentrations, or gene expression . In vitro enzyme activities and intracellular metabolite concentrations in aerobic batch cultures in bioreactors of the minimal glycolysis (mg) strain and a congenic reference strain . (a) average values of four independent culture replicates of the in vitro activities for the glycolytic enzymes in the mg strain (white bars) and the prototrophic reference strain cen.pk113-7d (black bars). Hxk, hexokinase / glucokinase; pgi, phosphoglucose isomerase; pfk, phosphofructokinase; fbpa, fructose - bisphosphate aldolase; tpi, triose - phosphate isomerase; gapdh, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphoglycerate kinase; gpm, phosphoglycerate mutase; eno, enolase; pyk, pyruvate kinase; pdc, pyruvate decarboxylase; adh, alcohol dehydrogenase . (b) intracellular glycolytic metabolite profiles of the mg strain (open circles) and of cen.pk113-7d (close circles) from aerobic batch cultures . Average values from two independent culture replicates are shown, and the average deviations of the mean are indicated by error bars . The vertical orange dotted line indicates the time at which glucose was depleted . If during evolution of s. cerevisiae, its glycolytic paralogs have evolved different roles through sub- or neofunctionalization, their deletion may only cause an observable phenotype under specific growth conditions . The growth rates of the mg and reference strains were therefore compared under a wide range of selected growth conditions . During fast growth in shake flasks on complex (yeast extract - peptone - glucose [ypd]) medium (fig . 4), the glycolytic pathway operates at rates closer to its maximum capacity than during growth at 30c on synthetic medium (60, 85). However, no difference in specific growth rates between the mg and reference strains was observed under these conditions . The mg strain also showed the same growth rate as the reference strain at high temperature (37c) (fig . Growth of the minimal glycolysis (mg) strain and a congenic reference strain under different growth conditions in shake flasks . (a) specific growth rates of s. cerevisiae strains mg (white bars) and cen.pk113-7d (reference strain, black bars). Smg, synthetic medium with glucose; smetoh, synthetic medium with ethanol as the carbon source; ypd, complex medium with glucose as the carbon source . The labels (b) growth, measured as change in the culture's optical density at 660 nm (od660), of the mg strain (open circles) and reference strain (closed circles) during carbon source switches . All data represent the average and average deviation of the mean from two independent culture replicates . Absence of the minor glycolytic paralogs did not affect growth of the mg strain on ethanol (fig . 2a and 4a). These results supported the conclusion from the aerobic bioreactor batch cultures that efficient gluconeogenesis does not require any of the minor paralogs (as has previously been proposed for adh2). While minor glycolytic paralogs apparently do not contribute to maximum specific growth rates on individual carbon sources, they might be involved in substrate transitions . For instance, pyk2, the glucose - repressed and fructose-1,6-bisphosphate - insensitive paralog of pyk1 (21), has been proposed to prevent futile cycling resulting from simultaneous operation of glycolysis and gluconeogenesis during transitions between fermentable and nonfermentable carbon sources (87, 88). However, no significant impact of the deletion of minor glycolytic paralogs was observed during transitions from glucose to ethanol and back to glucose (fig . 4b). To investigate possible phenotypes of the mg strain under a wider range of environmental conditions, growth on solid medium 5p to w), and at high concentrations of lithium and sodium ions, to which strains from the cen.pk lineage are hypersensitive (89) (fig . 5h to l). Only growth with glycerol as a carbon source indicated a slightly reduced growth rate of the mg strain . However, this phenotype was not observed during growth on liquid medium with glycerol (see fig . S3 in the supplemental material) and may therefore have been caused by a contaminating substrate in the agar used for the plate experiments . Growth of the minimal glycolysis (mg) strain and a congenic reference strain on different solid media . Serial dilutions of cell suspensions of the mg strain and of the reference strain cen.pk113-7d were plated on agar media with 24 different compositions . The conditions included osmotic stress (a to c), different phs (d to f), oxidative stress (g), three different salts (h to o), six different carbon sources (p to w), and complex medium (x). With the exception of panel x (ypd), all plates contained synthetic medium (sm). All plates contained 2% glucose, with the exception of plates with different carbon sources, which contained galactose (2%), maltose (2%), sucrose (2%), ethanol (3%, vol / vol), or glycerol (6%) and plates with low (0.03 m [5%]) and high (0.3 m [50%]) concentrations of glucose . Colonies of reference and mg strains are denoted by black bands and orange bands, respectively, at the bottom of each panel . Additional growth conditions tested on solid media were chosen based on information from the literature (74, 90) on sub- or neofunctionalization of specific deleted glycolytic paralogs . In addition to their enolase activity, eno1 and eno2 have both been implicated in vacuolar assembly, but in the absence of eno1, eno2 is able to support both functions (76). In the vacuole and the atpase associated with it, disruption causes a deleterious phenotype in s. cerevisiae when cultivated under alkaline conditions (91). As expected, the mg strain grew normally at ph 7.5, thereby indicating the absence of major vacuolar malfunction (fig . Tdh3 and tdh2, the two minor isoenzymes of glyceraldehyde-3-phosphate dehydrogenase, have been proposed to protect s. cerevisiae against oxidative stress because of their different levels of thiolation (92). However, exposure to oxidative stress by growth on hydrogen peroxide - containing plates (fig . 5 g) showed that the absence of tdh2 did not affect the oxidative stress resistance of the mg strain . Sub- and neofunctionalization have resulted in a different glucose - dependent regulation of glk1/hxk1 and pyk2 and in different regulatory properties of the proteins that they encode from those encoded by hxk2 and pyk1, respectively (21, 93). However, growth of the mg strain at low, intermediate, and high glucose concentrations was not impaired relative to that of the reference strain (fig . Finally, pdc6, which encodes a pyruvate decarboxylase isoenzyme with a low cysteine and methionine content, is specifically induced under sulfur limitation (94). Cultivation in the presence of cadmium increases abundance of pdc6 (9496); since cells require a high level of glutathione production for detoxification, sulfur amino acids are then used for this process and are less available for protein synthesis . Despite the absence of pdc6, the mg strain grew as well as the reference strain in the presence of cadmium . Deletion of 13 of the 27 glycolytic paralogs in s. cerevisiae yielded a minimal glycolysis (mg) yeast strain whose most spectacular characteristic was the absence of any pronounced phenotype under a wide range of laboratory growth conditions . One of the hypotheses that has been proposed to explain retention of functionally redundant paralogs during evolution is a contribution to gene dosage and, thereby, to the capacity of the pathway or process in which they operate (97, 98). The high glycolytic rates in anaerobic cultures of the mg strain (18 mmol glucose per g of dry biomass per h) did not significantly differ from those of wild - type strains (99) (fig . 2). This result argues against gene dosage effects as a means for fixing minor glycolytic paralogs in the yeast genome . Instead, our observations indicate that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of s. cerevisiae and essential for many of its industrial applications (100, 101). It might be argued that the gene dosage hypothesis does apply to phosphofructokinase, as deletion of either pfk1 or pfk2 substantially reduces enzyme activity and fitness (7981) (see fig . However, the presence of pfk1 and pfk2 in a hetero - octameric complex can also be seen as a case of neofunctionalization, in which two paralogs have been fixed by an acquired mutual dependency . The near - wild - type growth kinetics of the mg strain in aerobic and anaerobic cultures is difficult to reconcile with the hypothesis that duplication of glycolytic genes during the wgd event played a major role in increasing its glycolytic capacity or in causing the phenomenon of aerobic fermentation (the crabtree effect) (20). Our inability to identify a phenotype after deletion of all minor paralogs of glycolytic genes in s. cerevisiae does not imply that such a phenotype does not exist . The range of conditions tested represents an infinitesimal fraction of the environmental conditions to which s. cerevisiae may have been exposed to in its evolutionary history . The absence of a clear phenotype under standard laboratory conditions makes the mg strain an even more interesting platform for future high - throughput studies to investigate its phenotype under more conditions . Other characteristic features of s. cerevisiae like tolerance to high ethanol concentrations or growth at high gravity are interesting conditions to test, as well as the robustness of the mg strain under nonstandard conditions, such as sporulation, starvation, severe calorie restriction, and dynamic nutrient supply regimens . However, guessing the environmental factors that have conferred an evolutionary advantage to strains carrying gene duplications presents a formidable challenge, more particularly because the real niche for s. cerevisiae is still a matter of debate, to the extent that s. cerevisiae may be considered a generalist that does not necessarily favor a specific type of environment (102). For further functional analysis studies of the minor glycolytic paralogs, the set of intermediate strains used for construction of the mg strains (see table s3 in the supplemental material) can be used to rapidly identify which minor paralog or paralogs contribute to any newly identified phenotypes . Moreover, the sensitivity of fitness analyses can be improved for example, by competitive cultivation of wild - type and mg strains in mixed cultures . In s. cerevisiae, overrepresentation of paralogous gene sets is not unique for glycolysis (23 of 27 glycolytic genes are paralogs, corresponding to 85%) but also occurs for metabolic genes in general . Of all metabolic genes, 44% are paralogs (98), while this percentage is only 5% for the entire yeast genome (5, 17). Currently, a large research effort is under way to enable design and assembly of entirely synthetic yeast genomes (36). For the design of compact, functional synthetic genomes, it will be highly interesting to investigate whether our results on yeast glycolysis can be extrapolated to minor paralogs of genes encoding key enzymes in other central metabolic pathways . At present, predictions of the outcome of such experiments the clear impact of minor paralogs of transaldolase and transketolase on pentose fermentation kinetics by engineered s. cerevisiae (103) is a clear example that, at least under some conditions, minor paralogs can make a clear contribution to metabolic flux . The deletion of all minor glycolytic paralogs had a surprisingly small effect on the yeast transcriptome in aerobic, glucose - grown batch cultures . This result is in marked contrast with several studies in which deletion of individual major glycolytic paralogs led to transcriptional upregulation of its minor paralogs . Upregulation of minor paralogs is the upregulation of pdc5 upon deletion of pdc1 (104). Such an asymmetric cross - regulation is consistent with the backup theory for fixation of duplicated genes (17), which postulates that (minor) paralogs provide a buffer against deleterious mutations . Although upregulation of minor paralogs and even repair of a null mutation in a major glycolytic paralog by recombination with a minor paralog have been demonstrated (pdc1/pdc5) (104), the general evolutionary significance of the backup theory is still a matter of debate (98, 105). The selective advantages responsible for the overrepresentation of paralogous genes among the structural genes of yeast glycolysis therefore remain an intriguing conundrum . As the mg strain was constructed before the advent of crispr - cas9 technology (106), its construction involved 16 consecutive rounds of transformation and 11 marker recycling steps . A previous study (107) in which all 20 hexose transporter (hxt) genes in s. cerevisiae were deleted by repeated rounds of transformation and marker recycling resulted in massive genomic rearrangements, which were recently explained from repeated use of the loxp system for marker recovery (120). Repeated use of the loxp system enables recombination across loxp sites that are left in the genome after marker removal (108). Strain is mainly used as an excellent platform for functional analysis of individual transporter strains (107, 109117), in which role its chromosomal rearrangements are hardly relevant . While the mg strain similarly offers an interesting platform for functional analysis of (heterologous) glycolytic genes by one - step gene replacement, we designed and constructed it with the specific aim of building a robust platform for quantitative studies in systems biology . By using ura3 and amdsym (53) as counterselectable, recyclable marker genes, furthermore, interference of auxotrophies in the interpretation of quantitative growth studies (57) was avoided by making the mg strain prototrophic . The availability of a well - defined yeast platform with a minimal complement of glycolytic enzymes should provide clear advantages for quantitative modeling of the kinetics and regulation of glycolysis, as it eliminates the intrinsic uncertainties caused by the simultaneous, context - dependent expression of different isoenzymes . In view of the important role of glucose transport in the kinetics of yeast glycolysis, we are currently constructing mg variants with a single hexose transporter . In addition to providing a relevant test bed for mathematical modeling of glycolysis, the mg strain provides an interesting, simplified starting point for laboratory studies on yeast glycolysis . Previous studies have indicated that long - term laboratory evolution can have a major impact on glycolytic genes and their expression (59, 118). Comparison of evolution of the mg strain with that of strains that carry a full complement of glycolytic genes provides an interesting starting point for studies on the impact of gene duplication on evolutionary flexibility of a ubiquitous central metabolic pathway.
This retrospective study was approved by the institutional review board of our hospital (2014 - 09 - 037). Between november 2011 and october 2014, 245 patients in the icu underwent bedside pdt with a ciaglia blue rhino percutaneous tracheostomy set (cook medical, bloomington, in, usa) due to prolonged endotracheal tube insertion . Pdt was not performed in patients aged under 18 years, those with any pulsation palpated over the tracheostomy site, patients with a history of surgery or radiotherapy in the cervical region, and those with coagulopathy (increased prothrombin time, inr> 2). The first four procedures were performed by two physicians and one nurse using the standard technique, with one physician operating the bronchoscope while the other performed pdt . The next 55 procedures were performed by one physician and one nurse, with the physician using the bronchoscope only to confirm and evaluate the proper depth for the endotracheal tube before performing pdt using the simplified method described below . The remaining 186 procedures were performed using the simplified technique after repositioning the endotracheal tube at the predetermined depth (17 cm for males and 15 cm for females at the incisor). All pdts were performed by a single physician and one nurse, both of whom fully understood the entire procedure . The nurse administered sedatives and analgesics, as well as repositioning the endotracheal tube . Except for patients with limited neck extension, a small pillow was placed under the patient's shoulders to slightly extend the neck . Transverse markings were made at the cricothyroid membrane and 2.0 cm toward the sternal notch (fig . Was also made in the midline of the trachea by holding the trachea between the thumb and index finger . The middle of the vertical mark intersected with the transverse mark 2.0 cm below the cricothyroid membrane (fig . If the cricothyroid membrane could not be palpated, ultrasonography was used to identify the anatomic structures . The cricoid cartilage and tracheal rings were identified by placing the linear probe 45 degrees from the sagittal plane, and the area between the second and third tracheal rings was marked with a skin marker (figs . The linear probe was positioned vertical to the trachea to confirm its midline (figs . Fio2 was increased to 1.0, and 57 cmh2o positive end - expiratory pressure was applied to all patients receiving ventilator care . In patients who were receiving oxygen supplementation via a hydro - trach t - piece, the pdt site was disinfected with povidone iodine solution and a sterile drape was used before subcutaneously injecting 2% lidocaine . A vertical skin incision (1.5 cm) was made through the midline vertical marking, and the endotracheal tube was withdrawn until the tube depth was 17 cm for males and 15 cm for females (fig . 1d) using a 5 cm - introducer needle, included in the cook ciaglia blue rhino set, and proper needle position was confirmed by air regurgitation with a syringe containing normal saline . The remaining pdt procedure was performed using the previous standard method with a #7 tracheostomy tube (twist, tracoe, germany). A #8 tube was used only in patients in whom bronchoscopic lavage was planned (about 10 patients). However, all 3 patients were receiving either extracorporeal membrane oxygenation or continuous renal replacement therapy and had an abnormal coagulation status . Bleeding in all three long - term (> 7 days) follow - up in 150 patients showed no late complications and no deaths related to pdt . Although use of a fob during pdt has been recommended for precise positioning and to avoid complications, bronchoscopy itself can cause complications . Also, an additional physician is required and additional costs may result from damage to the fob . Thus, several studies have assessed the safety of pdt without a fob, including technical modifications and simplified procedures . Direct visualization of the larynx with a laryngoscope confirmed adequate endotracheal tube repositioning depth prior to pdt, while direct or indirect palpation of the trachea confirmed the proper tracheostomy site . Although these simplified techniques have been shown to be safe, they have not been widely adopted . This study assessed the safety of a simplified pdt technique using the ciaglia blue rhino, by repositioning the existing endotracheal tube at a pre - determined depth and determining proper pdt site by superficial palpation . Early complications of pdt include loss of airway, bleeding, conversion to open tracheostomy and death, with later complications including tracheostomy occlusion, tracheal stenosis and granulation . A retrospective study comparing early and late complications of pdt, with or without fob assistance, using the ciaglia blue rhino kit in 243 patients found no between group differences in early and late complications, but one patient experienced cardiac arrest during the use of a fob due to loss of airway . In this study, only 3 patients experienced bleeding after pdt, with all 3 receiving extracorporeal membrane oxygenation or continuous renal replacement therapy and having an abnormal coagulation status . However, we were unable to assess long - term complications in patients who were transferred to other facilities . Precise positioning is critical when performing pdt, thus requiring use of a fob or direct palpation of the trachea . The biggest disadvantage of our modified percutaneous method was the inability to confirm the pdt site . In five patients, where the underlying structures could not be adequately palpated another disadvantage is the lack of precise endotracheal tube repositioning, which can be achieved with a laryngoscope . Although a fob was used to determine the proper tube depth in 55 patients, the needle and guidewire entered the murphy eye of the tube twice . Pdt was successfully performed by retracting the tube an additional 12 cm in both cases . Although none of these patients experienced endotracheal cuff failure due to needle puncture, accidental cuff puncture may occur . Moreover, in contrast to subcutaneous dissection, physicians using our modified technique may be unable to identify vessels between the skin and trachea . Ultrasonography, which was used when anatomical structures were not readily identifiable, also has the advantage of screening such blood vessels . Since all pdts were performed at a single center by two physicians, and the proper endotracheal tube depth was evaluated in a korean population, it may be difficult to generalize the safety of our simplified technique . Another limitation was the absence of patients in which pdt is believed to be difficult, such as patients with severe obesity . Although studies have suggested that pdt is safe in obese patients, use of additional equipment, such as a fob or ultrasonograph, has been recommended . We have not performed pdt in morbidly obese patients who may also require extra - long tracheostomy tubes . Currently, therefore, a fob should be used when performing pdt in difficult situations, such as in patients with morbid obesity, anatomic alterations or neck stiffness . In conclusion, our results show that pdt with the ciaglia blue rhino technique can be safely performed without a bronchoscope and blunt dissection . This simplified technique is a relatively safe procedure that can be especially useful in situations of limited equipment and medical personnel.
We report here five newly sequenced and assembled penicillium genomes, which we compared with previously available data [1619]. The full dataset included the genome sequences of ten penicillium species, six of which are either used as industrial inocula for cheese making (penicillium roqueforti and penicillium camemberti) or occur as contaminants in cheeses (figure 1; table s1). Camemberti is only found in cheese and is thought to include a single clonal lineage originating from selection programs at the end of the 19 century from the blue - gray cheese molds used at that time, i.e., penicillium biforme and penicillium fuscoglaucum [20, 21]. By contrast, p. roqueforti also occurs in habitats other than cheese, such as silage or wood, and displays substantial genetic diversity [22, 23]. For reconstructing a rooted phylogeny of these ten penicillium species, we used four aspergillus species as an outgroup . We concatenated alignments of 3,089 single - copy genes shared by at least ten species and reconstructed a fully resolved and well - supported maximum - likelihood phylogeny (figures 1a and 1b). We used this rooted phylogeny to investigate the occurrence of horizontal gene transfers (hgts) between penicillium species . As hgts (also known as xenology) result in incongruences between gene genealogies and species trees, we compared all individual gene genealogies with the species tree . For this goal, we used the notung software [2527] to infer the number of duplication, loss, and hgt events that reconciled the gene genealogies with the species tree . Notung is conservative regarding the inference of hgts because it tests their temporal feasibility, assumes that hgts occur with a low probability, and forces the poorly supported nodes to follow the species tree . Only orthologous groups with at least one homolog in at least eight genomes were analyzed, further rendering our estimates of hgts a lower bound . Notung inferred the highest number of hgts relative to branch length in the clade encompassing p. camemberti, p. biforme, and their common ancestor (figure 1b). 8 of the 21 horizontally acquired genes detected in p. roqueforti were inferred to come from p. camemberti, p. biforme, or their common ancestor, indicating recent transfers from species sharing the same ecological niche (figure s1). Only five of these eight genes could be assigned putative functions, i.e., a protein kinase, two transcription factors, a cation transporter, and an integrase - like protein . Cation transport seems particularly relevant for growth in cheeses as several ions are limiting in this medium (e.g., iron ions), and ph drastically drops during cheese maturation . Another line of evidence for the abundance of hgts in penicillium fungi came from the finding of multiple large genomic islands that were almost 100% identical at the nucleotidic level between distant species, while being absent from closely related species (figure 1a). The only substitutions detected in these genomic islands corresponded to repeat - induced point mutations, i.e., c - to - t transitions induced by a specific fungal defense mechanism against transposable elements (tes) that can substitute multiple base pairs in a single meiosis . In p. roqueforti, for example, seven genomic islands larger than 10 kb and displaying above 97% nucleotide identity with multiple other species were found . Such a high level of identity suggests that these genomic islands correspond to recent horizontally transferred regions (htrs), although they could alternatively be recent introgressions . Two lines of evidence, however, support the htr hypothesis rather than introgression: (1) the presence of several of these regions at non - homologous locations in the different penicillium genomes (figure s2;) and (2) the low mean genome sequence identity between the penicillium species sharing these regions, being less than 90%, an identity level at which no successful interspecific crosses have ever been reported in fungi . Interestingly, these htrs were flanked in p. roqueforti by copies of tes from a particular family that were rare elsewhere in the genomes (figures 1a and s3), the i non - ltr retrotransposons . The other abundant tes (e.g., mariner dna transposons and copia retrotransposons) were in contrast scattered in the genomes (figure 1a). The genes present in these genomic islands of high sequence similarity partially overlapped with the hgts detected by notung . In p. roqueforti for example, 17% of the hgts inferred by notung were located in the seven large htrs (figure 1a). The fact that not all genes in htrs were detected by notung mainly results from the filter of this analysis where we used only orthologous groups with homologs in at least eight species . Further, 11% of the inferred hgts in p. roqueforti clustered within 50 kb of the htrs (figure 1a), suggesting that these genomic regions may be prone to integrate foreign dna . This is consistent with the previous finding that the genomic region where wallaby is inserted in p. roqueforti carries other species - specific genes in each of p. camemberti, p. rubens, and p. roqueforti . The identification of multiple very recent htrs, with almost 100% identity in multiple species (figure 1a), together with notung inferences of horizontally transferred genes occurring also elsewhere in the genomes (at least 104 in total among penicillium fungi), indicates that hgts occur frequently among penicillium . The clustering of i elements in the flanking regions of htrs suggests that they may be involved in the mechanism of horizontal transfers . It has been shown that tes can pass across species boundaries and that they promote genomic rearrangements and recombination [3234]. The capacity for mycelia fusions may also facilitate the exchange of genetic material in fungi . Five of the seven large htrs detected in p. roqueforti were shared between penicillium strains isolated from cheese, p. camemberti carrying four of them (figure 1a). Wallaby carries a gene encoding an antifungal protein, known for inhibiting the growth of competitors . The second largest htr was found at terminal edges of scaffolds and was therefore named cheesyter . This 80-kb region, found as a single block in all the genomes studied, carried 37 putative genes, among which two had relevant putative functions for adaptation to cheese, i.e., lactose permease and beta - galactosidase (figure 2a). Lactose is present during the first few days of cheese maturation, and it acts as a primary carbon source, being rapidly consumed by lactic acid bacteria . The two lactose metabolism genes present in cheesyter were among the most strongly expressed in p. camemberti during the first step of cheese rind maturation, during which lactose is available (figure 2b; table s2; supplemental experimental procedures), indicating a role in the use of the cheese substrate . We investigated the presence of cheesyter and wallaby by pcr in 416 strains from 65 fungal species from various environments (figure 1c; table s3; supplemental experimental procedures). The presence of the transfers was found highly significantly associated with dairy environment both among species (= 55.7; degrees of freedom [df] = 1; p value = 8.571e14) and among strains within species (2 = 45.2; df = 1, p value = 1.774e11). Amplicons were actually obtained only for penicillium species that are frequently isolated in the dairy environment, with the only exception of p. rubens, the penicillin - producer fungus, in which 16 strains out of 20 carried either one of the two htrs . The cheesyter and wallaby fragments obtained by pcr showed zero substitution among all strains from all species, including synonymous sites and non - coding regions, as previously found for wallaby . This result confirms that the presence of cheesyter and wallaby is not ancestral in penicillium species and that these genomic islands have instead been acquired very recently . This also indicates that the two htrs have spread in several species through recent selective sweeps . P. roqueforti was found polymorphic for the presence of wallaby and cheesyter, with all tested strains carrying either both of these regions or neither of them (table s3). Within p. roqueforti, these regions were present only in strains isolated from the cheese environment, suggesting a role in adaptation to the cheese environment . We therefore investigated whether strains carrying wallaby and cheesyter showed higher fitness in terms of growth on cheese substrate or for competitiveness . We set up three experiments, focusing on p. roqueforti, because a large collection of strains was available, isolated from various environments, and including strains carrying both wallaby and cheesyter (hereafter named w+c+) and strains lacking them (hereafter named wc). We first compared the growth of 50 p. roqueforti strains on a cheese medium and on a minimal medium (26 w+c+ and 24 wc; table s4, tab a). Neither the presence of wallaby and cheesyter, as a main effect independent of the medium, nor the origin of the strain (i.e., cheese versus other environments) significantly influenced the growth of p. roqueforti (table s1). By contrast, the effect on growth of the medium and its interaction with the presence of the two genomic islands were significant (figure 3; table s1): w+c+ strains had a growth advantage on cheese medium but a slower growth on minimal medium . Second, we investigated whether p. roqueforti strains carrying wallaby and cheesyter had a higher ability to exclude competitors . We measured the growth of three fungal strains belonging to species commonly found in cheese but lacking wallaby and cheesyter (p. nalgiovense fm193, p. biforme lcp05529, and geotrichum candidum fm074) on plates covered with lawns of p. roqueforti either w+c+ (n = 11) or wc (n = 12) strains (table s4, tab b). These experiments were carried out on minimal, cheese, and malt agar media . No difference in growth was detected for the yeast g. candidum between lawns of w+c+ or wc p. roqueforti strains (table s1). By contrast, w+c+ p. roqueforti strains significantly impaired the growth of the two penicillium challengers on the cheese and malt media (figure 3b). This was not the case on the minimal medium: the interaction between the presence of the transfers and the medium was significant (table s1). Using the same experimental design, we then investigated the effect of the two genomic islands when present in the challengers . For this goal, we inoculated on p. roqueforti lawns (w+c+, n = 2, or wc, n = 2), on cheese medium, different strains of species displaying a polymorphism in the wallaby and/or cheesyter presence (table s4, tab c). We used as challengers different strains of p. camemberti (w+c, n = 1, or w+c+, n = 3), p. biforme (wc, n = 2, or w+c+, n = 2), and p. rubens (wc, n = 1, or w+c,, we found that the p. roqueforti lawns significantly inhibited the growth of challengers and significantly more so when the p. roqueforti lawn carried wallaby and cheesyter . Interestingly, the presence of either cheesyter or wallaby in the challengers allowed better growth on wc p. roqueforti lawns while neither had significant effect on the growth on w+c+ p. roqueforti lawns (table s1). Third, we investigated competition among p. roqueforti strains carrying (w+c+, n = 8) or lacking (wc, n = 11) wallaby and cheesyter . We grew p. roqueforti strains on cheese medium as pairwise face - to - face confrontations, and we measured the deviations from symmetrical growth (table s4, tab d; figure 3c; supplemental experimental procedures). For the w+c+ versus w+c+ confrontations, the mean growth deviation from a boundary in the exact middle of the petri dish was not significantly different from zero (t test, t = 1.5; df = 36; p value = 0.14). Similar results were obtained for the wc versus wc confrontations (t test, t = 0.25; df = 70; p value = 0.80). For the w+c+ versus wc confrontations, deviations were measured by taking the w+c+ strain as the focal strain; the mean growth deviation was significantly different from zero and positive, the w+c+ strains thus growing farther than the wc strain (t test, t = 12.32; df = 90; p value <0.0001). The mean deviations were significantly higher in the wc versus w+c+ confrontations than in the w+c+ versus w+c+ or wc versus wc confrontations (tukey - kramer test, p value <0.0001), while the means between these two latter were not significantly different . This experiment shows that the competitive advantage of w+c+ strains against wc strains also holds within the species p. roqueforti . Altogether, these experimental results strongly support the existence of fitness advantages for the penicillium strains carrying the horizontally transferred genomic islands, both in the use of cheese substrate and in competition with fungal competitors . The two cheese species studied here underwent parallel adaptation to the cheese medium, and this involved the transfers of identical regions across species boundaries . Hgt events have been reported in fungi [35, 38, 39], particularly in environments created by humans, in domesticated yeasts and fungal pathogens of crops [10, 40, 41]. The extent and timing of gene transfers and the number of species having received the same htrs are here particularly striking . Furthermore, we provide experimental evidence of fitness advantages for strains carrying these htrs on a human - made medium . These findings altogether are potentially useful for guiding modern strain improvement programs . Indeed, together with the protocol for inducing sex in p. roqueforti [22, 42], the identification here of several key candidate genes important for cheese metabolism and competition may allow further selecting interesting traits for cheese industry using the great genetic variability present in p. roqueforti strains without wallaby or cheesyter . In addition, our results suggest that caution is required concerning the introduction of genes into microorganisms, as these genes could readily be transferred to other species in the food environment . Indeed, the rapid spread of wallaby and cheesyter into many species of the dairy environment, even when occurring only as contaminants, indicates that transgenes may readily cross species boundaries in the food chain . Finally, the findings here of rapid adaptation through frequent horizontal gene transfers among distant species under selection in novel, human - made media contribute to our understanding of the evolutionary genomic mechanisms allowing rapid adaptation to environmental changes in eukaryotes . ; investigation, a.b ., m.l .- v ., r.c.r.d.l.v ., and j.r.
Orthodontic anchorage is a basic procedure necessary for a successful treatment . For this reason, clinicians seek to better understand various orthodontic devices, such as headgear, transpalatal arch, lingual arch, and nance's button . These devices allow a certain degree of movement of the anchorage unit and are dependent, to varying degrees, on patient compliance . In severe cases or in cases of non - cooperative patients, treatment could be compromised . Recently, skeletal anchorage has increased in popularity due to the benefit of direct transfer of the point of anchorage from the teeth to the skeleton . However, since these mini - implants have been used in orthodontics, a small displacement during treatment has been detected . The mini - implant displacement allows contact between the screw and the dental root, causing damage to the root or the periodontal ligament, potentially leading to mobility or loss of the mini - implant . Unfortunately, there is not much information available on the displacement or adverse effects of mini - implants . Recently, hsieh, et al . (2008) found that even endosseous titanium implants may not necessarily serve as a rigid orthodontic anchorage at all force levels . (2004) investigated cephalometric tracings and found a noteworthy extrusion and tipping forward of mini - implants after en - masse retraction of anterior teeth . Recently, tomographic studies have evaluated the position of mini - implants in both jaws, though without relating them to longitudinal control . To the best of our knowledge, general dentistry and orthodontics constantly use radiographs with the main purpose of assessing the validity of treatment methods . Zanda (2007) described a new film holder for occlusal film that showed effective reproducibility of occlusal radiographic images in dry skulls (figure 1), making it possible to take an occlusal radiographic sequence at different times . (2005) used dry human skulls to analyze the reliability in determining tooth position in posterior - anterior films . Using markers inserted in the bands of molars and premolars from hyrax expanders allow identifying each one in each side on posterior - anterior films . Occlusal x - ray film holder and skull positioned for standardized occlusal radiograph the purpose of the present study was to evaluate the reliability and accuracy of a radiographic analysis method for identifying the location of mini - implants inserted in the posterior region of the maxilla in dry human skulls . Three human skulls were obtained from the anatomy department of the dental school of the university center of maranho, brazil . All skulls had preserved maxilla and mandible and sufficient teeth on the posterior area of the maxilla for the successful placement of mini - implants . The cortical bone condition and the maxillary interadicular space were evaluated using radiographic images between the second premolars and the first molars . According to the same technical criteria used for patients, six self - drilling mini - implants (1.6x1x7 mm; osas, dewimed, tuttlingen, germany) were placed between the second premolars and the first molars at a 45 angle relative to the tooth's long axis . Once the study began, the main purpose was to test the reliability of different operators for the proposed method in this research . Therefore, clinical operators with various levels of professional experience were invited to take the occlusal radiographs . Three operators were selected: an orthodontist, an implantodontist and an undergraduate dental student . Each operator performed three occlusal radiographs taken at three intervals of 15 days in each of the three skulls . The set of three radiographs of each skull was identified externally according to an identification number for the skull, the operator and the time . A total of 27 randomized occlusal radiographs were obtained using insight io-41 film (size 5.7 cm x 7.6 cm; kodak) with 1.5 s exposure time at 10 ma and 70 kv . The occlusal radiographs were taken with a correct alignment between the cylinder localizer from the x - ray machine and the ring from the occlusal radiographic position self - drilling film . Each operator took the occlusal radiograph for each of the skulls with the occlusal films inserted in the zanda (2007) film holder and fit the film in more number of posterior teeth of the maxilla . All radiographs and a ruler with same lateral size were digitized using a scanner (be@rpaw 2400ta plus, scanner a4) (figure 2). The radiographs were measured using macbiophotonics imagej software originally applied in biologic researches for microscopic image capture . After the mini - implant positions, the points were defined and measured using a cartesian coordinate system (figure 3). The cartesian system was composed by the following measures: occlusal radiographs with mini - implants placed occlusal radiographs and measurements line a: a straight line traced over the maximum number point of the midpalatal suture; line b: a line perpendicular to the intersection of line a at the virtual point representative of the incisive foramen; distance xd: for the mini - implant on the right side, the minimum distance from the head of the mini - implant to line a; distance yd: for the mini - implant on the right side, the minimum distance from the head of the mini - implant to line b; distance x' d: for the mini - implant on the right side, the minimum distance from the tip of the mini - implant to line a; distance y' d: for the mini - implant on the right side, the minimum distance from the tip of the mini - implant to line b; distance xe: for the mini - implant on the left side, the minimum distance from the head of the mini - implant to line a; distance ye: for the mini - implant on the left side, the minimum distance from the head of the mini - implant to line b; distance x' e: for the mini - implant on the left side, the minimum distance from the tip of the mini - implant to line a; distance y' e: for the mini - implant on the left side, the minimum distance from the tip of the mini - implant to line b. the means and standard deviations of the distances on each side were calculated considering the operator and the skull (table 1). The intra - operator reliability for the measurements was estimated using the interclass correlation coefficient (icc). Analysis of variance (anova) was used to test the effect of different image times and different operators . Mean and standard deviation (sd) for each measurement and operator (op) statistical analysis indicated a significantly high, positive correlation between the maxillary occlusal radiography taken by the different operators (table 2). Intra - operator reliability for mini - implant measurements expressed as interclass correlation coefficient (icc) (n=3) the icc test verified the measurements of each variable made by each operator on each skull that localize the head and the tip of the mini - implant (x, y, x' and y'). Intra - operator correlations were above 0.87 for all the operators and each of the skulls (table 2). This value seems to indicate a similar variation, indicating an excellent reproducibility through a positive and high correlation . In terms of the effect of time, the operator and its interactions showed no statistically significant differences in any of the variables analyzed by anova (table 3). The goal of the present study was to test the reliability and accuracy of the radiographic occlusal position developed by zanda (2007) used as a device to verify the stability of posteroanteriorly and lateromedially positioned orthodontic mini - implants . The hypothesis was that this method can be used as a replicate for maxillary occlusal radiographs for location of mini - implant placement in the posterior region of the maxilla . Since the introduction of the cephalostat as a standard for taking cephalometric radiographs, the cephalostat has been traditionally used to determine the direction and amount of skeletal and dental changes . Several devices have also been developed and improved with longitudinal research in orthodontic patients . Some studies have reported that film holders and markers of anatomic structures contribute to the improvement in radiographic images . Also creating more information about the position of structures during orthodontic treatments . Based on these presumptions, our study used a model of an occlusal x - ray film holder to obtain the most uniform radiographic series . Additionally, the selected anatomic structures used as references to determinate the measurements were chosen because they are known to be stable during treatment . This study showed that the use of the occlusal x - ray film holder is sufficiently accurate for investigations of mini - implant stability and an alternative to cephalometric analyses for determinate mini - implant displacement . Furthermore, the numerical information on the displacement of the mini - implants can be correlated with the amount of dental movement determined by the retraction . (2004) reported that mini - implants do not remain stable at forces higher than 400 g. these findings suggest that studies are necessary to quantify mini - implant displacement caused by treatment, especially by means of the innumerable variables related to the use of this transitional anchorage device . Our findings show that future clinical studies will be able to evaluate the direction of the displacement of the mini - implants . Also, different types of this skeletal anchorage device could be compared considering the thickness, the design and the length with the suffered displacement . Computed tomography (ct) scans are not routinely used to improve the quality of craniofacial measurements in orthodontics . The present data prove that the method presented here is valid and can be performed at a much lower cost compared to ct . This means that clinicians and researchers can better understand this innovative modality of anchorage using an easily attainable and low - cost material . Another important benefit of this method is a lower exposure of patients to radiation to detect the stability of the mini - implants . We believe that clinical studies on this topic are very important for further acknowledgment of transitory anchorage device . However, there is a great deal of responsibility in deciding to prescribe radiographs, especially ct scans; it is complicated and depends on the effects on the orthodontic therapy management . Human skulls were used for safety reasons due to the potential exposure of patients to ionizing radiation . The adopted model simulates similar conditions used for patients, with a reduced x - ray exposure time . Additionally, the anatomic structures used to obtain all measurement tracings are easily identified, as well as their presence in the same plane and height as the indicated inserted mini - implant in the posterior maxillary area ., we believe that the selected areas are not influenced by soft tissue thickness and do not compromise the visualization of the anatomic structures used and the installed mini - implants . The number of dry skulls used in this type of study has revealed variations in other studies . The number of skulls used in the present study was sufficient to verify the influence of individual anatomic variations in the statistical analysis . It should be noted that we opted for a research design that did not evaluate the anatomic aspects in relation to accuracy and reliability . The aim of the study was to analyze operator's reproducibility on separate occasions . The intraclass correlation proved that, with the skull change and, consequently, the occurrence of anatomic variations, a high and significant correlation was found among the different measurements of each skull . The agreement in the operators' measurements (table 3) shows that this method is useful for research and for clinical evaluation . For clinical use, it is indicated to attach the film to the film holder (figure 1) and after adaption inside of the mouth, ask for the patient to occlude . Only with the dental occlusion is possible to achieve stability to use this intraoral radiographic technique . Zanda (2007) found variations in the reliability of measurements far from the occlusal plane, whereas the present study showed no statistically significant differences . In the present study, this reproducibility was found because the mini - implants and the anatomic structures used the same plane into the maxilla, both are at the same tall of palatal plane . Variables describing the position of the head and the tip of the mini - implant were in the same plane . Also, the midpalatal suture was chosen as a reliable reference for the determination of a measurement for each variable . It should be mentioned that the method presented here only identifies mini - implant localization on the posterior region of the maxilla . This implies that, in future studies, an additional use for this method in another site of the maxilla or mandible may be the identification of a more reliable anatomic structure for each studied region . The results of the present study demonstrate that this radiographic method was highly reproducible among operators and accurate for the identification of measurements . Also, no differences were observed based on the time and the operator using this method . These results support the use of this standardized method for occlusal radiographs for mini - implant identification on the posterior maxilla.
Heart transplantation continues to provide patients with end - stage heart disease with extended survival with a half - life of 9.3 years between 2000 and june 2008 . . However, despite substantial advancements in immunosuppression, patients continue to be at significant risk for allograft rejection early after cardiac transplantation . The two recognized forms of allograft rejection are acute cellular rejection and antibody - mediated rejection (amr). While acute cellular rejection has historically been the most common cause of allograft dysfunction, amr has only recently become widely accepted . During the 2004 international society of heart and lung transplantation (ishlt), cellular rejection grades were revised and amr was formally defined . In april 2010, a publication from the ishlt consensus conference assessed the status of amr in heart transplantation and a pathologic grading scale was devised . Despite the advent of new technology, such as gene expression profiling and echocardiograms, endomyocardial biopsy remains the standard for detecting rejection . To minimize the risk of a false negative, multiple specimens (usually 35) are obtained from 3 different sites . Though rare, false negatives do exist either through sampling error, artifact, quilty lesions, or pathology misread . Prior to the 2010 consensus conference, hemodynamic compromise, in the absence of acute cellular rejection, was termed biopsy - negative rejection . Although the prevalence of the so - called biopsy - negative rejection has declined with the clinical diagnosis of amr, there are incidences of patients who present with cardiac dysfunction (left ventricular ejection fraction, lvef 45%) but have no biopsy findings of cellular or antibody - mediated rejection . Since the outcome of patients with bnr (which includes cardiac dysfunction) has not been well established, we sought to review the characteristics and treatment response of patients who have developed bnr after heart transplantation . We retrospectively reviewed our cohort of patients who underwent heart transplantation between 2002 and 2012 and found 12 cases of bnr in 11 heart transplant patients, as defined by patients presenting with cardiac dysfunction, characterized by lvef 45%, and who had no biopsy findings of cellular rejection or amr . As severe infections are also known to cause left ventricular dysfunction, patients with a clear clinical picture of sepsis with fever, increase in white blood count, or positive cultures were excluded . Continuous variable was presented as mean standard deviation while categorical variable is presented as percentages . We identified 11 patients who underwent heart transplantation between 2002 and 2012 and were treated for bnr . None of the bnr patients had previous blood transfusion or previous transplant . 5 (45%) patients had a previous vad placement and 1 (9%) patient was african american . The baseline immunosuppression medications are as follows: 5 patients (45%) underwent induction therapy with antithymocyte globulin (atg), 9 patients (82%) initiated with tacrolimus and mycophenolate mofetil, 1 patient (9%) initiated with cyclosporine and mycophenolate mofetil, and 1 patient (9%) initiated with cyclosporine and azathioprine . 2 patients switched to cyclosporine from tacrolimus because of reduced seizure threshold; 1 patient switched to a renal sparing protocol of sirolimus with mycophenolate mofetil because of renal function concerns . The 12 cases of bnr presented with an average lvef of 34% 10% and 11 (92%) treated cases occurred during the first - year after transplant (table 3). The mean time to the first incidence of bnr is 7.8 7.5 months and no patient had treated rejection before onset of bnr except for the patient at the second onset . Among the 106 biopsies that have been done before bnr, 24 (23%) demonstrated low grade acute cellular rejection (1r, 1a, or 1b) and 1 (0.9%) showed suspicious antibody - mediated rejection (amr 1). De novo circulating antibodies developed in 3 cases (in 3 patients) and 1 of them had donor - specific antibodies . 4 out of the 12 cases presented with lvef 35% and presented with heart failure symptoms . Of these 4 heart failure cases, 2 required inotropic support both received intravenous (iv) methylprednisolone (500 mg1000 mg per day for 3 days), atg (125150 mg per day for 35 days), and iv immunoglobulin (ivig, 70 g per day for 3 days), followed by oral corticosteroids (prednisone 80100 mg per day bolus and taper). For the remaining 2 patients who presented with heart failure symptoms but did not necessitate inotropes, 1 was treated with atg and iv methylprednisolone followed by prednisone bolus and taper and the other was given iv methylprednisolone followed by prednisone only . For the 8 out of 12 cases without heart failure symptoms, 7 were treated with only high dose oral corticosteroids . Overall, 7 cases (58%) (in 7 patients) favorably resulted with return to normal left ventricular function (lvef of 57% 6%) within a mean of 14 10 days from the initial negative biopsy . Of these 7 patients, one expired approximately 6 months after the date of the normalized lvef and another one expired approximately 4 months after the date of normalized lvef . The remaining 5 cases (in 5 patients, including 1 patient's second case of bnr) maintained persistent left ventricular dysfunction beyond 90 days . A flow chart of treatment and effect could be found in figure 1 . In summary, the lvef status at 90 days is as follows: 5 cases (in 5 patients, including 1 case as second bnr on a patient) had persistent lv dysfunction, and 7 cases (in 7 patients) experienced normalized lvef . As our understanding of the biological mechanisms underlying cardiac allograft rejection increases, the number of undiagnosed rejection decreases . The concept of bnr has evolved from its initial definition as hemodynamic compromise in the absence of acute cellular rejection to its current definition of patients presenting with cardiac dysfunction in the absence of biopsy findings of cellular rejection or amr . Amr has changed from being suggested as bnr to its own ishlt biopsy grading scale . However, even though the majority of rejection episodes of unclear etiology have now been resolved to be cases of amr, there are still cases where the biopsy findings are inconsistent with the clinical prognosis, that is, patients with a decrease in lvef with no biopsy findings of rejection, cellular, or humoral . Due to the inconsistencies of endomyocardial biopsies, the existence of bnr is questioned . This argument is furthered by the fact that prior to the 2010 amr consensus conference, 1020% of cardiac allograft recipients were diagnosed with bnr when in actuality the majority of patients most likely experienced amr . This brings up the question of whether bnr should exist as a separate category of rejection or is merely a by - product of the problems inherent with endomyocardial biopsies . In one case study, sampling error or nonuniformity of histopathologic changes resulted in a false - negative biopsy . In this case study, a heart transplant recipient with repeatedly unremarkable endomyocardial biopsies and a negative evaluation for humoral rejection was found to have severe subepicardial myocyte necrosis with classic cellular rejection in the subsequent autopsy . This is contrary to other studies which have found that rejection is evenly distributed throughout the right ventricular endomyocardium . Although endomyocardial biopsy is the primary resource to diagnose acute rejections in all the cases discussed in this study, there are a few noninvasive diagnostics that have been demonstrated to be helpful in diagnosing acute allograft rejection after heart transplantation . In one multicenter clinical trial, pham et al . Reported that using gene - expression profiling to monitor rejection for patients 6 months after heart transplantation was not associated with increased risk of serious adverse outcomes and could reduce the need for biopsy . In two pilot studies, wu et al . Applied cardiac magnetic resonance (cmr) in vivo to detect immune - cell infiltration at sites of rejection by monitoring macrophages . The investigators subsequently developed a functional index from local strain analysis and proved it to be correlated with rejection grades [8, 9]. Although clinical applications have not been implemented, cmr was demonstrated to be capable of providing the rejection status of whole - heart perspective, and thus might be a potential tool of optimizing diagnosis of bnr . Another potential tool is speckle - tracking 2-dimensional strain echocardiography (2dse). By using rat cardiac transplantation models, pieper et al . Demonstrated that 2dse was able to differentiate myocardial function between rejection in allografts and nonrejection in isografts . Perhaps bnr is another form of amr but due to the newness of amr and the lack of a complete understanding of amr, no definite conclusions can be made . Despite the ishlt revised biopsy rejection scale, this revised definition of bnr has been noted to result in a decrease in 3-year subsequent survival, lower subsequent freedom from cardiac allograft vasculopathy (cav), and a decrease in freedom from nonfatal major adverse cardiac events (nf - mace). Previous studies characterized bnr as occurring on average 43 38 months following transplantation while the data collected in our study indicates that the time to the first incidence of bnr is 7.8 7.5 months . The mechanism associated with bnr unfortunately not too many reports in the field looked at the treatment protocols for bnr . In our single center experience, who patients presented with heart failure symptoms were given more aggressive treatment, that is, atg, iv methylprednisolone, and ivig while patients without heart failure symptoms were mostly given prednisone bolus and taper only . As bnr is an infrequent phenomenon, of which there is considerable debate regarding its validity, further work needs to be instigated on whether there is a correlation between different factors and bnr episodes . Overall, from this set of data, it is inconclusive as to whether or not there are any characteristics of bnr that differs from other types of rejections . A point of interest in this study is the patient who experienced two episodes of bnr . Due to the small sample size, it is uncertain whether having one episode of bnr increases the risk of recurrent bnr episodes . During the first bnr episode, in contrast, it took the patient 6 months to experience a return to normalized lvef (51%) during the second bnr episode . For the first episode of bnr, there was a favorable return to normal cardiac function following treatment with oral corticosteroids bolus and taper . In the case of the second episode of bnr, ivig was given instead which led to an increase in lvef, although it took a longer time (6 months) for the patient to experience a return to the range of normalized lvef . Prior to experiencing normalized lvef the patient's lvef remained in the 40% range, fluctuating between 43% and 48% . The repeat bnr patient was the only african americans in this study and did not experience normalized lvef at 90-day after second onset . African americans have been reported to have a higher risk of rejection after transplant compared to any other race due to their unique metabolism in regard to immunosuppression [14, 15]. A possible immunosuppression regimen with respect to bnr could require more aggressive treatment, employing the use of atg and ivig in addition to steroids . Potential cause for this second onset of bnr could be the combined factor of this patient not receiving an adequate treatment for the second bnr and the ethnicity of the patient . As cases of bnr tend to respond favorably to appropriate rejection therapy 2 (17%) of 12 bnr cases required inotropes and 7 (58%) bnr cases without heart failure symptoms were treated with oral steroid bolus and taper . Seven of 12 (58%) treated bnr cases resulted in a return to a normal lvef of 57% 6% within a mean of 14 10 days from the initial negative biopsy . Due to the small sample size, it is uncertain whether bnr should be considered as a new category in the biopsy grading scale or if it is merely difficult to diagnose based on endomyocardial biopsies alone . If it is the former, then mechanism needs to be elucidated . If it is the latter, then perhaps other methods for detecting rejection need to be considered . Bnr is a rare phenomenon (12 cases of bnr in a 10-year period) and can be a severe form of rejection in that there is cardiac dysfunction . However, this type of rejection is not apparent on biopsies due to either lack of knowledge regarding further types of rejection or other factors . Characteristics of these cases of rejection are described above with most cases responding favorablly to rejection therapy.
Are frequently present in the water systems of large buildings, and exposure to these bacteria occurs therefore regularly . Nonetheless, legionnaires' diseases (lds), the most severe form of illness due to legionella spp ., seem to be a rare outcome of exposure . This has been underpinned by outbreak investigations suggesting that only 0.15% of persons exposed to legionella develops ld . Most legionella infections may be subclinical or result in an influenza - like illness (pontiac fever). In particular, subclinical infections may be common among individuals with regular exposure to legionella [1, 2]. In an outbreak of ld at a floral show, antibody levels were higher in exposed but asymptomatic exhibitors than in the general population . Health complaints differed by the workplace locations of the exhibitors but were largely independent of antibody levels . Although legionella has been detected by culture in up to 70% of water samples from hospitals' water distribution systems [48], and nosocomial ld is a well - known problem, little is known about rates of legionella infections in communities and workplaces . The aim of the present cross - sectional study was to analyse antibody levels among hospital workers with known exposure to legionella and to determine the correlation between antibodies to legionella and self - reported symptoms compatible with legionella infection . Furthermore, we examined domestic and other environmental risk factors for seropositivity among the hospital workers . The study was undertaken at a 643-bed acute - care hospital providing both general and specialised hospital care . The hospital blocks include both new and old buildings up to a hundred years old . There have been no cooling towers functioning in the hospital area since 2001 . Before 2003 there were 21 separate hot water systems with blind ends in every system . From 1998 to 2003 all hot water tanks were removed and replaced by heat exchangers . As part of measures for reducing the risk of legionella infection at the hospital, the temperature of the outgoing hot water is maintained at least 60c; whereas the circulating temperature and the temperatures at the most remote points - of - use are at least 50c . Once a week, the temperature is increased to 6770c in about three hours . There is no routine monitoring of the temperatures of the water in the pipes or at the points - of - use . In spite of these precautions, six nosocomial ld cases from five departments were reported at the hospital between 1999 and 2005 . The hospital has guidelines for the prevention of ld among susceptible patients, including recommendations to avoid exposure to aerosols and to use sterile water for drinking purposes, and so forth . Water samples from the hospital were analysed for viable legionella at statens serum institut within two days of sampling . The results were recorded as the highest number of colonies confirmed as legionella (cfu / litre). From each water sample with growth of legionella, one to five colonies were selected and tested by legionella latex test (oxoid dr0800, basingstoke, uk), by this method the isolates were divided into l. pneumophila serogroup 1, l. pneumophila serogroup 214, and legionella spp . The lowest count of legionella that reliably can be detected by this method is 100 cfu / litre . In the period 1999 to 2005, 230 waters samples were analysed, and 214 (93%) were positive for legionella spp . With counts up to 28 0000 cfu / litre . All departments included had positive water tests for l. pneumophila, and l. pneumophila sg 1 were found in all departments but one . The samples (74) taken in the year of the study, 2005, showed that all water distribution systems of the selected departments were positive for legionella with counts up to 18 000 cfu / litre . L. pneumophila sg 1 was present in 14% of the samples, sg 214 in 60% (l. pneumophila sg 3 in 19%), and in 1% of the samples legionella spp . A month before our study we tested representative showers at the departments and at the staff changing - rooms; three of four showers at the staff changing - rooms showed low levels of legionella spp . A total of 675 employees from nine different hospital departments were invited to participate in the study . The eligible employees had various risks of exposure, including showering patients, performing surgical hand wash, or using the shower of the hospital for personal purpose . All participants were asked to complete a questionnaire about self - reported health and relevant exposures during the past year . The questions about health status included a history of ailments such as influenza - like illness, pneumonia, common cold; health care seeking including hospitalisations and visits to general practitioners; absences from work due to illness; specific symptoms (cough, fever, malaise, stomach pain, shiver, diarrhoea, headache, myalgia, cold). Participants were requested to report symptoms only if they had persisted for at least two consecutive days in the previous year . The questions on occupational exposures included frequency and duration of showering patients, using a shower at the hospital for personal purpose, and frequency of performing surgical hand wash . Combined hospital exposure included any frequency of showering patients, self - showering, or surgical hand wash . The questions on nonoccupational exposures (reflecting potential environmental risk factors) included type of residence; residence built before 1970; district heating; presence of hot water tank; hot water tap - time (considered to be slow if not hot in 1/21 minute); closure of water distribution; closure of home; use of spa - bath; shower elsewhere than home; swimming pool; travel abroad; hotel stay in denmark; visit to a danish summer cottage; air - condition in private car . Socioeconomic variables were school - education, job skills, and family income . All questions concerned exposures during the previous year . Blood samples from hospital workers were analysed for antibodies to legionella by indirect immunofluorescence antibody test (ifat) with plate grown and heat inactivated l. pneumophila serogroup (sg) 1 to 6, l. micdadei and l. bozemanii as antigens . The assay is based on the well - characterised assay described by wilkinson et al ., which follows the guidelines from the centers for disease control and prevention (cdc). The in - house if test has recently been compared with commercial kits for detection of antibodies to l. pneumophila, and it was found that the in house test was at least as specific and sensitive as the commercial kits .the serum samples were titrated from 1: 64 and upwards to end - point titre . A titre of 1: 128 was used to define a positive antibody response to legionella . National laboratory test criteria for a confirmed diagnosis of legionella infection include a four - fold or greater rise in antibody titre to 1: 128 in ifat (seroconversion) to l. pneumophila sg 1, 3, or 6 . Seroconversion to other legionella antigens and positive titres (1: 256) to any legionella antigen are considered indicative of a recent or previous legionella infection . To compare the results obtained for health care workers with the general population, we analysed blood samples collected from 308 and 400 healthy blood donors living in the two towns of randers and vejle, as described previously . These towns are situated in the neighbour regions of the catchment area of the study hospital . In 2004, the incidences of notified ld in the two towns were 48 and 19 per million inhabitants, respectively . In 2004, the incidence of ld in the town of the study hospital was 17 per million which is within average incidence of ld in denmark . There was no difference in age, gender, or overall antibody distribution between the towns . Epi data (ver 3, odense, denmark) was used for data entry . Univariable analyses were performed with antibody status as the dependent variable; variables with p <.2 were added to the model . Based on this p - value, multiple logistic regression analyses were applied to determine associations with health status and risk factors, respectively, adjusted for age, gender, and current smoking . Variables of significance in the multiple analysis were reported with odds ratio (or) and 95% confidence interval (ci). The prevalence of seropositivity declined by age in a log - linear fashion and therefore age (in years) was fitted as a continuous variable . The study was approved by the regional scientific ethical committee (vn2005/7) and the danish data protection agency . The antibody titres 1: 128 for all serogroups were significantly higher in the hospital staff (45.1%) than in the donor population (22.9%) (or 3.41, 95% ci 2.444.77). There was no significant difference in antibody levels for l. pneumophila sg 1 (or 1.43, 95% ci 0.942.16). One person only from the hospital staff was positive to l. bozemanii, and none was positive to l. micdadei . The hospital staff had a mean age of 44 years (range 20 to 67 years) with a male / female ratio of 36/222 . There was no difference in mean age and range or smoking between the hospital staff and the healthy blood donor population, although nearly a quarter (22.5%) of the donors was smokers . Male / female ratio was differently, with 56.9% of the donors being males, however, among donors the antibody levels were independent of gender . In general, there were no marked differences in self - reported morbidity between seropositive and seronegative individuals . Persons with legionella antibodies tended to report more absence from work due to illness or having had a common cold than seronegative persons reported (table 1). Furthermore, persons who reported symptoms other than influenza - like symptoms had a lower risk of developing antibodies (or 0.34; 95% ci 0.120.99), but the numbers were small (table 1). Based on a p - value of <.2, multiple regression analysis of seropositives (titre 1: 128) revealed association with previous pneumonia (or 0.29; 95% ci 0.090.94) and current smoking (or 3.54; 95% 1.1011.49). Seropositives for sg 1 (titre 1: 128) were not associated with any of the health - related variables in multiple regression analyses . Furthermore, we constructed a symptom complex of influenza - like illness (cough, fever, malaise, stomach pain, shiver, diarrhoea, headache, myalgia, cold). There were no consistent association between legionella antibodies and a symptom complex of at least three (or 1.95; 95% ci 1.003.78), four (or 1.08; 95% ci 0.901.29), or five (or 0.99; 95% ci 0.841.17) symptoms of the complex of influenza - like illness with adjustment for age, gender, and current smoking . Finally, there were no associations between symptoms and exposures at the hospital (data not shown). Individuals taking showers in other places than home or having air - conditioning in private car had an increased risk of having antibodies (table 1). The multiple regression model (seropositives with titre 1: 128) of risk variables with p <.2 and gender, age, current smoking showed significant increase in antibodies with showering elsewhere than home (or 1.89; 95% ci 1.083.31), air - conditioning in car (or 1.99; 95% ci 1.153.35), and decreasing age (or 0.97; 95% ci (0.940.99). Multiple regression analysis of seropositivity to sg 1(titre 1: 128) showed increased antibody levels to sg 1 when having a hot water tank at home (or 4.49; 95% ci 1.5313.1) and by decreasing age (or 0.95; 95% ci 0.901.00) and decreased antibody levels when having had hotel stays in denmark (or 0.32; 95% ci 0.110.95). Further age analysis in age groups showed a lower prevalence in persons 50 years and above (or 0.79; 95% ci 0.650.95) compared with individuals below 50 years . Antibody levels were independent of hospital department and type of occupational exposure (data not shown). Thus, there were no significant differences in antibody level between staff members working with patients, showering patients, taking personal showers or doing surgical hand wash . There were no differences according to frequency of the exposure (almost daily to never). We found a higher prevalence of legionella antibodies (1: 128) in the hospital staff with continuous exposure from the water system than in blood donors being representative of the general health population . Antibody levels were not higher in members of the hospital staff at risk of frequent aerosol exposures from showers or surgical hand washing . We found no association between an influenza - like symptom complex and the presence of antibodies . The epidemiology of subclinical legionella infections is largely unknown, especially beyond the outbreak setting . However, outbreak investigations indicate that the antibody response in the healthy population declines with the distance to the source of the outbreak [13, 14]. An outbreak of pontiac fever indicated coherence between attack rate and distance to source too . Compared with other studies we found a high prevalence of seropositive individuals suggesting a high exposure and probably ongoing exposure at the hospital . This finding is consistent with an italian study of a healthy hospital staff which found a high prevalence of antibodies to l. pneumophila sg 114, but only a prevalence of 3.0% for l. pneumophila sg 16 which are the serogroups (especially l. pneumophila sg 1) most frequently reported causing disease . The distribution of the levels of legionella antibody shifts to the right (higher levels) with increasing exposure in an outbreak situation; a similar distribution may occur at the hospital setting with a probable higher exposure of the staff compared with the donor population . We found no specific sources of exposure at the hospital nor subgroups being at higher risk . This is in contrast to a study from italy where dental personnel had a higher risk of antibodies compared to other hospital workers, possibly due to dental staff being close to aerosols . Aerosols have been shown to be able to spread over a large area outdoors . We do not know if aerosols will disperse over large areas indoors, but this is conceivable . Surprisingly, a small fountain without obvious aerosols - generating capability was recently implicated as the source of an outbreak of ld . This corroborates that exposure to legionella arising from aerosol - generating sources at health care facilities may occur relatively far from the source . The hospital workers distance to aerosol sources at the hospital or their number of contacts with the sources had no influence on their antibody level . The questions about exposure and health were all about conditions in the previous year . The health symptoms are common, frequent, and probably not easy to remember . This poses the problem of recall bias, but recall bias will affect both groups equally as no one is aware of having antibodies . Self - reported exposure time and frequencies of exposure at work seem to be valid and useful . The association between types of symptoms and high antibody levels in some previous studies seems to be weak and inconsistent [1, 3, 20]. We found no symptoms related to high antibody level, even though single chance findings could be expected due to the large number of tested symptoms . Our study was limited by being based on the serological analysis of single serum samples; it is well known that antibodies to legionella can be detected months after an infection . Reliable serological diagnosis of a recent or current legionella infection can best be done by the detection of a seroconversion, which for our if test is defined as a fourfold rise in titre to at least 1: 128 . In addition, it can take two to several weeks before antibodies can be detected after the onset of symptoms or after exposure . A follow - up study of a staff cohort would have enabled us to detect both the changes in antibodies and the related symptoms . We do not know to what extent the seroprevalence varies in different populations, though we found hardly any variation between our two blood donor populations in the two different towns, one with an average incidence and one with an endemic high incidence of ld, respectively . We know that the incidence of ld in the study town was within the average incidence of ld in denmark, and we therefore assume that the overall prevalence of antibodies to legionella in the population in the town of our hospital was at the same level as the donor populations in the reference towns . An inverse relation between age and seroprevalence has not been demonstrated in other studies [14, 16]. We investigated the staff at a hospital with an ongoing high amount of legionella in the water system . We found that almost half of the staff had serological signs of legionella infection, but these antibodies could not be related to specific occupational exposures or symptoms . Although this indicates that the health implications for workers at health care facilities may be limited, we do not know the health risks if a virulent legionella invades the distribution system . Treatment and maintenance of water systems in healthcare to minimise the threat of legionella contamination following well - described methods should therefore be a standard procedure in order not only to minimise the risk of nosocomial ld but also to reduce occupational risks.
Dizziness has a high incidence worldwide, and the impact of vestibular disease on quality of life has been increasingly investigated . Many patients with dizziness restrict their daily activities and leisure in order to reduce the risk of onset of unpleasant and frightening symptoms, as well as to avoid the social embarrassment and stigma that these symptoms may cause1 . Benign paroxysmal positional vertigo (bppv) is a disease with a high incidence worldwide, and can be regarded as the most frequent vestibular disease2 . There are few studies correlating bppv and meniere's disease (md) and their combined influence on the quality of life of patients3 4 . Simultaneous diagnosis of bppv and md may worsen symptoms of dizziness and thus worsen the quality of life, because the discomfort caused by these symptoms can significantly change the ability of patients to perform their usual tasks4 . Among existing evaluation instruments, the dizziness handicap inventory (dhi) is an internationally validated instrument . This questionnaire assesses patients' perception of the effects of the disabling vertigo on their quality of life, including the physical, emotional, and functional aspects . It is important to assess the individual's condition at the beginning of treatment and to monitor the evolution of the disease course1 (chart 1). Legend: physical aspects - questions 1, 4, 8, 11, 13, 17 and 25; functional aspects - questions 3, 5, 6, 7, 12, 14, 16, 19 and 24, emotional aspects - questions 2, 9, 10, 15, 18, 20, 21, 22 and 23 . Each yes answer = 4 points; sometimes = 2 points; no = 0 points . Treatment options for vertigo patients include medications, vestibular rehabilitation (vr) and, more rarely, surgical procedures5 . Vr has been used as a strategy for the treatment of vestibular disorders because its goals are to provide security to the patient and integrate them into their social environment . Vr aims to promote visual stabilization, improve static and dynamic balance, decrease sensitivity during head movement, and improve the overall function of the patient via proprioceptive and vestibular visual stimulation maneuvers6 . The objective of this study was to evaluate the pre- and post - vr quality of life of patients with md - associated bppv by using the brazilian version of the dhi as an evaluation tool . The study was approved by the ethics in research on humans foundation regional university of blumenau (furb), under protocol 180/09 . The sample consisted of 12 patients of both genders with a diagnosis of meniere's disease associated with benign paroxysmal positional vertigo (bppv) of the posterior canal, who were seen at the ent clinic during the period from october to december 2009 . Inclusion criteria were as follows: clinical diagnosis of meniere's disease with complementary electrocochleographic findings, presence of nystagmus triggered by dix hill pike maneuvers and lateral placement test identified under the guidance of videonystagmography, no treatment initiation or maintenance during the period between surveys, agreement to undergo surgery to the ear, and agreement to signing a consent form . We excluded patients with signs and symptoms of vestibular central origin, those with musculoskeletal abnormalities that impair the performance of the maneuvers (diagnostic and therapeutic), individuals who began vestibular rehabilitation before completing the questionnaire, and patients undergoing vestibular rehabilitation lasting less than 5 sessions, considering the research period . The sessions were held once a week individually, with each session lasting 1 hour . Vr started with the canalith repositioning maneuver of epley, where the individual sitting on the table adopts a supine position and the therapist rotates the head to the side of the affected labyrinth . After a minute, the head is rotated to the opposite side and the patient is instructed to stay in the same position for another minute, returning to a seated position at the end . This was followed by the brandt and daroff habituation exercises . From the seated position on the table, the patient adopts a lateral recumbent position (first on the side that causes the dizziness), maintains the position for 30 seconds or until the dizziness passes, changes to a lateral seated position, waits for the same time period, and then lies on the opposite side and waits for 30 seconds . Each patient was taught the exercises and instructed to repeat them at home 1020 times a day . In addition, we performed the cawthorne - cooksey visual exercises consisting of up and down and side - to - side eye movements as well as extension of an arm and focusing on the finger as it approaches and moves away from the face . All movements were repeated 20 times, starting slowly and becoming progressively faster, with the patient sitting or lying down . In another exercise, with the individual sitting or standing, only the head moved forward and back, after a back and forth motion; these motions were first performed slowly and then rapidly, with eyes open and progressing to accomplishment of each movement and with eyes closed for 1 minute . The brazilian version of the dhi was completed by participants before undergoing the first session of vr and after the last treatment session . Subjects were instructed to mark the item that best demonstrated their perception of the influence of vertigo on their quality of life at the time, with doubts addressed by the researchers present during the survey . No, corresponding to 0 (zero), occasional presence of symptoms / problems was reported as sometimes, and was scored as 2 points, and the presence of severe symptoms / problems was marked the minimum score of the questionnaire was 0 (zero) and the maximum was 100 points, with 7 items referring to physical aspects, 9 to functional aspects, and 9 to emotional aspects7 . For data analysis, we first used the shapiro - wilk test of normality, proceeding with the wilcoxon test for comparisons between samples before and after vestibular rehabilitation, and the friedman test to compare the impact of physical, emotional, and functional scores after standardizing the values by calculating the percentage according to the maximum possible score to be obtained on each item . Spearman correlation was used to evaluate the relationship between aspects, the total score, and the characteristics of the sample test . All analyses were processed in 4.0 bioestat considering significance at p <0.05 . In the stipulated period for data collection, 12 individuals fulfilled the criteria for inclusion and exclusion . There were 4 (33.33%) men and 8 (66.66%) women, all caucasian, aged between 35 and 86 years (mean, 53.17 15.75 years). Three (25%) were less than 40 years old, 6 (50%) were aged between 40 and 59 years, and 3 (25%) were aged 60 years or more (table 1). There was a significant reduction in all dhi values after vr, independent of the aspect . The median final values were 0 (zero) for the physical aspect, 1 for the emotional aspect, and 1 for the functional aspect, demonstrating the favorable impact of vr on the quality of life of our patients (table 2). Generally, the score must be reduced by at least 18 after treatment to be indicative of benefit1 . In our series, 7 (58.33%) patients showed a reduction greater than 18 points, 5 (41.67%) did not show a reduction greater than 18 points, and 3 (25%) did not achieve a pre - vr score large enough to show such a reduction . In the evaluation of post - vr scores, 2 (16.6%) patients who had scores less than 18 pre - vr, despite showing an improvement in the aspects evaluated, did not show score reductions of the specified magnitude . P <0.01 when compared to before . To compare the effects on the different aspects, the raw score values were normalized as percentage values, with 100% indicating the maximum score in every aspect . Next, we compared the pre- and post - vr scores . In the preoperative evaluation, we noted that the diseases had a greater impact on physical and functional aspects than on the emotional aspect . Although none of the aspects showed a predominant effect of vr, the differences in the pre- and post - vr values showed that vr had the greatest influence on the physical aspect (table 3). P <0.05 when compared to emotional aspect in the first evaluation (pre - vr), the correlation between the physical and functional aspects was moderately positive, and that between the emotional and functional aspects was strongly positive, indicating the interrelationship of these factors on quality of life of the volunteers, i.e., the functional aspect influences and is influenced by physical and emotional aspects . However, analysis of the difference (pre - post) showed a weak positive correlation between the emotional and functional aspects (table 4). In the pre- and post - vr analyses, there was a lack of correlation between age and gender with the dhi scores, indicating that the results of the vr were not affected by these characteristics (table 5). It should be noted that the sample size did not allow generalization of the results; however, considering the results, the importance of vr is evident . In another study with 15 subjects who were diagnosed as having bppv associated with mnire's disease, the physical aspect was the most affected4, but there are few studies analyzing both conditions together and using vr for the treatment of this combination . Regarding gender, there was a predominance of women (75%) in the sample, which agrees with previous studies that found a prevalence of dizziness and bppv in 61.3% to 62.5% of female patients8 9 10 . Some authors suggest that the variations in natural hormones in women could be related to this increased incidence, but did not elucidate this theory11 . As for meniere's disease (md) alone, there are reports of an equal distribution between genders12 . The most prevalent age group in this study also coincides with the findings obtained by other authors, who indicated that 48% to 60% of subjects were between 41 and 60 years of age4 7 . Other studies have also reported the coexistence of these 2 diseases, with patients initially presenting symptoms of md followed by symptoms of bppv13 14 . One hypothesis for this relationship between the 2 diseases is the possible release of otoconia by damage to the utricle by endolymphatic hydrops and hypertension3 . The dhi - brazilian version was used to assess the impact of vr on the quality of life of patients and, based on the significant score decrease in most patients, it is argued that vr is favorable for these patients . The dizziness and other symptoms that often accompany vestibular disorders may manifest during social, familial, and professional activities, thus causing physical, financial, and psychological distress to the patients15 16 17 and necessitating interventions to eliminate or minimize these symptoms . Individuals end up restricting their head movements and activities to prevent vertigo, thereby imposing physical constraints that lead to functional loss and consequent emotional turmoil, which explains the damage caused by these disorders . We found that pre - vr, the greatest impact was noted on the physical and functional aspects while after vr, the greatest influence was noted on the physical aspect, despite the reduction of the values in the functional and emotional aspects as well . It was noted that after the improvement in physical symptoms, individuals were able to resume their routine activities that were previously restricted due to dizziness or fear of symptoms, and this improvement consequently improved functional and emotional aspects, which are dependent on the physical aspects . This result contrasts the results of a previous study18, where a sample of 6 patients, aged 4370 years, with complaints of dizziness and tinnitus showed no significant improvement in the functional and emotional aspects and no improvement in the physical aspect after vr . It should be noted that apart from vertigo, some individuals had age - related comorbidities (such as osteoarthritis and osteoporosis), which by itself has limited some of the emotional issues such as fear of high places and fear of being alone at home or going out unaccompanied . Therefore, it is likely that the answers to these questions were influenced by comorbidities, so it is not possible to separate them from the symptom of vertigo / dizziness . This situation indicates that the analysis of the emotional aspect should be undertaken with care, because if the individual is unable to perform some physical activity for problems not related to dizziness, even after the improvement of vertigo, he / she will be unable to do the same . The correlation between the physical and functional aspects and between emotional and functional aspects in pre - vr assessments demonstrates the interrelationship of these factors with the quality of life of individuals, i.e., the functional aspect influences and is influenced by the physical and emotional aspects . However, this effect appears to be greater when the symptoms are exacerbated by vertigo, since after vr, no such correlation was observed . The relationship between the functional and physical aspects is also highlighted in other studies, where the similar scores for these aspects were explained by the fact that in bppv, onset of symptoms is closely related to certain positions or head movements, the relevant questions for which are contained in both aspects of ihl - brazilian version19 20 . Similarly, studies involving individuals with md associated with higher scores on the physical and functional aspects to the chronic nature of the disease with clinical manifestations floating, recurrent and lasting, that can compromise not only physical, but also the functionality of these patients4 21 . In another study with 6 patients aged 4370 years, the analysis of the dhi questionnaire after vr showed that patients experienced an improvement in functional and emotional aspects, although the difference was not significant18 . Furthermore, the relationship of the functional to the emotional aspects can be understood by the rationale that when there is improved functionality, the individual returns to his / her daily habits, accomplishing all movements restricted before the onset of vertigo . Thus, the individual becomes more confident with regard to leaving the house without company and can simply raise the head or turn over in bed without being afraid of triggering symptoms . The results of vr were not affected by age and/or gender, similar to previous studies that indicate no relationship between improvement in patients with vertigo and after vr with these variables20 22 . In this context, the geriatric population responds to treatment as well as a younger population, but most of the elderly patients require a greater number of treatment sessions to achieve the same result as the young23 . Moreover, other studies describe that age is not necessarily associated with loss of independence in activities of daily living, and therefore, it is not possible to relate the age - diminished ability with greater independence or improvement in vertigo after vestibular alteration24 . On the other hand, dizziness has a more detrimental effect on quality of life of elderly than in younger adults, according to studies with these populations25 26 27 28 . Regarding gender, some authors report that the variable is not related to any advantage or disadvantage in relation to response to treatment7, a fact verified by the results presented here . As individuals in the sample were out of the crisis period during the questionnaire analysis and underwent vr, the results discussed are restricted to perception of symptoms by individuals on a daily basis, whereas in vertigo, the symptoms are exacerbated . Finally, we would like to point out the importance of multidisciplinary medical and physiotherapy in the diagnosis and treatment to achieve greater security and adherence to this treatment modality . The results obtained through the dhi brazilian version show that the quality of life of patients with bppv associated with md improved after 5 sessions of vr in all aspects analyzed . We noticed that the diseases had a greater impact on the physical and functional aspects of quality of life before vr, with the highest rate of improvement noted in the perception of physical appearance after the therapy . Although the sample consisted of a small number of patients, the results obtained with vr were surprising and further studies need to be developed, especially involving the 2 pathologies . Moreover, the vr was well accepted by volunteers who showed increased enthusiasm and confidence with the evolution of treatment and reduction of symptoms.
Coronary heart disease (chd) is the cause of 17.1 million deaths per year throughout the world (1). Thus, chd is today the largest single contributor to global mortality and will continue to dominate mortality trends in the future . It has been reported that the majority of deaths (39%) in low- and middle - income countries under the age of 70 years are due to chd (2). It has been estimated that mi is 50% higher in south asians than in white people in the uk (5). Pakistan is a developing south asian country with a population of over 187 million (6). The majority of the population of pakistan (67.5%) live in rural areas and bear the greatest burden of heart disease (7). The framingham study (1961, cited in kannel) reported that obesity, hypertension, smoking, diabetes mellitus, and hypercholesterolemia were the major risk factors for the onset of chd (8). Another study showed that the high prevalence of mi risk factors in pakistan with more than 30% of the population over 45 years of age is affected by this disease (2). Punjab is the most developed and populated province accounting for more than 45% of the entire population of pakistan (9). However, there is a paucity of data on the estimates of chd risk factor burden or of its control status in punjab, pakistan . Moreover, only a little information on mi risk factors has been reported in peshawar and rahim yar khan, pakistan (7). The objectives of the present study were to collect epidemiological data on mi in north punjab and to evaluate the environmental risk factors responsible for mi with a view to lessening the disease burden through modifications in lifestyle . This population - based study was carried out in six regions of north punjab, pakistan . The urban population from lahore and islamabad and the rural population from faisalabad, gujranwala, gujrat, and sialkot a total of 515 mi patients were included in this study and divided into 250 urban and 265 rural mi patients . Data were collected by trained qualified staff from the patients admitted to coronary care units (ccu) or equivalent cardiology wards of the participating hospitals . This study was conducted in accordance with the good clinical practice (gcp) guidelines (declaration of helsinki). The study protocol was approved by the ethics committee of advanced studies and research (ref #aeg - a - ad-25 - 17/zool/09). Written informed consent was obtained from all the participants . The inclusion criteria were comprised of mi as the principal diagnosis and admission via the ccu and emergency wards . The diagnosis of mi was based on severe chest pain of 30 minutes duration, characteristic electrocardiographic (ecg) patterns of mi, and significant elevation in cardiac enzymes such as creatine kinase - myocardial band (ck - mb). The analysis was focused on identifying the socioeconomic status (i.e. Income and education), lifestyle (i.e. Smoking, leisure time, and physical activity), dietary pattern, personal and family history of mi, and risk factors such as blood pressure, diabetes, high - density lipoprotein cholesterol (hdl - c), and low - density lipoprotein cholesterol (ldl - c). At this point, arterial pressure was measured three times using a sphygmomanometer . According to the european society of hypertension / european society of cardiology (esh / esc) criteria, cases with systolic blood pressure> 140 mmhg and diastolic blood pressure> 90 mmhg as well as those with a previous history of hypertension and current consumption of antihypertensive medication for blood pressure control were considered hypertensive . Weight and height were also measured in order to calculate the body mass index (bmi) using the formula: weight (in kilograms) divided by the square of height (in meters). Cases with a bmi between 25 and 29 were considered overweight and subjects with a bmi were considered obese, while those with a total cholesterol level 200 mg / dl were considered hypercolesterolemics . Fasting blood samples (3 ml - 5 ml) of all the participants were collected by trained staff and were sent to the laboratories of the participating hospitals for lipid profile and blood sugar measurement . Subjects with a fasting glucose level 126 mg / dl were considered hyperglycemic, and those with a history of diabetes mellitus and using glucose - lowering medicines were considered diabetic . Disease treatment cost was expressed in pakistani rupees (rs). Patients not willing to sign the consent form and those with previous illnesses associated with hepatic and renal failure, human immunodeficiency virus (hiv), or cancer were excluded from the study . A descriptive analysis was conducted, and the study variables were checked for normal distribution . The kruskal wallis, mann - whitney u, and chi - square tests were utilized in order to check associations between the different variables . A two - sample two - tailed t - test (pooled variances) for a p value <0.05 was considered statistically significant . This population - based study was carried out in six regions of north punjab, pakistan . The urban population from lahore and islamabad and the rural population from faisalabad, gujranwala, gujrat, and sialkot a total of 515 mi patients were included in this study and divided into 250 urban and 265 rural mi patients . Data were collected by trained qualified staff from the patients admitted to coronary care units (ccu) or equivalent cardiology wards of the participating hospitals . This study was conducted in accordance with the good clinical practice (gcp) guidelines (declaration of helsinki). The study protocol was approved by the ethics committee of advanced studies and research (ref #aeg - a - ad-25 - 17/zool/09). Written informed consent was obtained from all the participants . The inclusion criteria were comprised of mi as the principal diagnosis and admission via the ccu and emergency wards . The diagnosis of mi was based on severe chest pain of 30 minutes duration, characteristic electrocardiographic (ecg) patterns of mi, and significant elevation in cardiac enzymes such as creatine kinase - myocardial band (ck - mb). The analysis was focused on identifying the socioeconomic status (i.e. Income and education), lifestyle (i.e. Smoking, leisure time, and physical activity), dietary pattern, personal and family history of mi, and risk factors such as blood pressure, diabetes, high - density lipoprotein cholesterol (hdl - c), and low - density lipoprotein cholesterol (ldl - c). At this point, arterial pressure was measured three times using a sphygmomanometer . According to the european society of hypertension / european society of cardiology (esh / esc) criteria, cases with systolic blood pressure> 140 mmhg and diastolic blood pressure> 90 mmhg as well as those with a previous history of hypertension and current consumption of antihypertensive medication for blood pressure control weight and height were also measured in order to calculate the body mass index (bmi) using the formula: weight (in kilograms) divided by the square of height (in meters). Cases with a bmi between 25 and 29 were considered overweight and subjects with a bmi were considered obese, while those with a total cholesterol level 200 mg / dl were considered hypercolesterolemics . Fasting blood samples (3 ml - 5 ml) of all the participants were collected by trained staff and were sent to the laboratories of the participating hospitals for lipid profile and blood sugar measurement . Subjects with a fasting glucose level 126 mg / dl were considered hyperglycemic, and those with a history of diabetes mellitus and using glucose - lowering medicines were considered diabetic . Disease treatment cost was expressed in pakistani rupees (rs). Patients not willing to sign the consent form and those with previous illnesses associated with hepatic and renal failure, human immunodeficiency virus (hiv), or cancer were excluded from the study . A descriptive analysis was conducted, and the study variables were checked for normal distribution . The kruskal wallis, mann - whitney u, and chi - square tests were utilized in order to check associations between the different variables . A two - sample two - tailed t - test (pooled variances) for a p value <0.05 was considered statistically significant . The total number of the male patients was 356 (69.13%) as compared to 159 (30.88%) females in this study . The mean age of the participants from the rural areas was 56.8 14.34 and from the urban areas was 55.7 11.26 with a range of 20 - 80 years, while 53.79% of the patients were in the range of 41 - 60 years of age (p = 0.270) among the rural and urban populations in both genders . Tables 1 and 2 show that among the urban and rural populations, mi was much more common in the males than in the females (p = 0.015), which was highly statistically significant . However, the male mi patients were younger than their female counterparts . The relative difference regarding the mi prevalence between the sexes was greater among the younger subjects of up to 60 years old and smaller among the subjects of more than 60 years of age . The demographic characteristics of the male and female subjects are summarized in table 1 . (%) or mean sd . Rural, rural population (faisalabad, gujranwala, gujrat, and sialkot); urban, urban population (lahore and islamabad). The body mass index was considered normal at 25 and overweight at> 25 for both men and women according to the world health organization . Abbreviations; nstemi, non - st - segment elevation myocardial infarction; and stemi, st - segment elevation myocardial infarction . Smoking was more common (table 2) in the mi patients in the rural areas (33%) than those in the urban areas (28%) (p = 0.205). Twenty - six percent of the selected mi cases had hyperlipidemia (cholesterol level 200 mg / dl). The prevalence of hypertension was 36% as compared to diabetes (19.4%) in the mi patients (table 2). As is evident in tables 3 and 4, the hdl - c level reduced, while the ldl - c level and hypertension rose with age . As is shown in figure 1, the patients with a first - degree relative positive family history experienced mi at a younger age at a bmi 25 . Abbreviations: bmi, body mass index; hdl, high - density lipoprotein; ldl, low - density lipoprotein; nstemi, non - st - segment elevation myocardial infarction; and stemi, st - segment elevation myocardial infarction . Abbreviations; bmi, body mass index; hdl, high - density lipoprotein; ldl, low - density lipoprotein; nstemi, non - st - segment elevation myocardial infarction; and stemi, st - segment elevation myocardial infarction . Figure 2 depicts the relationship between the monthly income and the overall cost (i.e. Ecg, computerized tomography (ct scan), angiography, surgery, and medication) of the mi patients (p = 0.917). According to the kruskal test, however, the mean monthly cost of medicines and physicians fees was 2381.132 rs (24.24 usd). Smoking was more common (table 2) in the mi patients in the rural areas (33%) than those in the urban areas (28%) (p = 0.205). Twenty - six percent of the selected mi cases had hyperlipidemia (cholesterol level 200 mg / dl). The prevalence of hypertension was 36% as compared to diabetes (19.4%) in the mi patients (table 2). As is evident in tables 3 and 4, the hdl - c level reduced, while the ldl - c level and hypertension rose with age . As is shown in figure 1, the patients with a first - degree relative positive family history experienced mi at a younger age at a bmi 25 . Abbreviations: bmi, body mass index; hdl, high - density lipoprotein; ldl, low - density lipoprotein; nstemi, non - st - segment elevation myocardial infarction; and stemi, st - segment elevation myocardial infarction . Abbreviations; bmi, body mass index; hdl, high - density lipoprotein; ldl, low - density lipoprotein; nstemi, non - st - segment elevation myocardial infarction; and stemi, st - segment elevation figure 2 depicts the relationship between the monthly income and the overall cost (i.e. Ecg, computerized tomography (ct scan), angiography, surgery, and medication) of the mi patients (p = 0.917). According to the kruskal test, however, the mean monthly cost of medicines and physicians fees was 2381.132 rs (24.24 usd). Risk factors associated with chd in the pakistani population have been relatively insufficiently studied (10). Nevertheless, a thorough knowledge of these factors in the different regions of punjab, pakistan, is of a great importance in order to develop national health strategies for their control . The current study found that chd and its risk factors such as hypertension, diabetes, and obesity are on the increase in our country . Our data revealed that the male mi patients from the rural and urban areas of punjab, pakistan, were relatively young (41 - 60 years). Gender difference was also common among our study population (p = 0.015), which chimes in with previous studies reporting that middle - aged men have 2 to 5 times higher risk of mi than women (11, 12). Women of young age are protected from the risk of chd (13), and hormone replacement therapy reduces the risk of heart disease in postmenopausal women (14). Estrogen is thought to be a major contributor to premenopausal women s tendency to have normal blood pressure, higher levels of hdl - c, and lower triglyceride levels compared to men (15, 16). In the current study, a high prevalence of overweight was found among the patients from the urban areas (p = 0.0041) as compared to those from the rural areas . This may be due to less physical inactivity and unhealthy dietary practice, encompassing the high consumption of saturated fats and refined carbohydrates as well as the low consumption of fruits and vegetables among the residents of urban areas . Our data on the bmi demonstrated that the patients with a positive family history of mi suffered heart disease even at a lower bmi 25, which may be due to consanguineous marriages in pakistan (18). Our results showed that a 56% st - elevation myocardial infarction (stemi) continues to be a major public health concern in pakistan . 19) also underlined stemi, by comparison with non - st - elevation myocardial infarction (nstemi), as a major issue in pakistan . In our study, sedentary lifestyle (70%) and smoking (60%) were reported predominantly among the male mi patients . Smoking deteriorates hdl - c (14), raises the blood pressure, and releases free radicals which are injurious to heart s health . Wu et al . (20) reported that the cessation of smoking can reduce the risk of heart disease by 65% . Faisal et al . (21) reported a 26% prevalence rate of sedentary lifestyle among patients with chd in peshawar, pakistan . Furthermore, smokers of all age groups have 2 - 3 times higher death rates than non - smokers (22). It has also been proven by an animal study that physical exercise improves the left ventricular function (23). In the present study, hypertension (36%) emerged as the most important risk factor for the onset of mi, and its incidence was much higher among the patients from the urban areas than among those from the rural regions . This finding is concordant with the results of a previous study reporting that hypertension is much higher in pakistan than in the other south asian countries (24). Hypertension is one of the most important risk factors responsible for atherosclerotic events (25). Similar to serum cholesterol, the blood pressure also tends to increase with age, and more prominently in women than in men (26). In our study, hyperlipidemia and diabetes were detected in 26% and 19.4% of our mi patients, respectively . According to the interheart study, hyperlipidemia is a significant risk factor for chd in south asia (27). Type ii diabetes is a major public health issue in pakistan (28) and is an important cause of cardiovascular disease (29, 30). The data available in the literature show a more frequent occurrence of other chd risk factors among diabetic patients (31). An earlier study reported that plasma ldl - c increases by about 40% between 20 and 60 years of age (32). Low- and middle - income individuals are faced with a great socioeconomic burden in the pursuit of their treatments . The diagnosis of heart disease via ecg, ct scan, echocardiography, and angiography is costly, while the main burden of disease faced by the patient is at the time of surgery . Our data showed that the mean calculated cost per patient for surgery was 600000 rs (6108.19 usd), which sometimes drastically affects the financial capacity of the patient . Indeed, patients have to consult their physicians every month, and medicines as well as physicians fees are expensive . In our study, the mean calculated monthly cost of medicines and physicians fees per patient was 2381.132 rs (24.24 usd). Even low - income patients are compelled to pay these high expenses, which may contribute to hypertension . One - third of pakistani people live in poverty and as such cannot afford the increasing burden of such a costly disease . Our data demonstrated that the mi patients living in the urban areas, in comparison with those living in the rural regions, had a high level of mi risk factors (i.e. Physical inactivity, hypertension, and diabetes) and should, therefore, be treated as a high - risk group for prevention and treatment . Moreover, this study demonstrated that effective risk factor control is still poor in punjab, pakistan . Accordingly, public health programs, comprising lifestyle interventions and different pharmacological therapies, should be implemented in order to reduce the risk of chd in pakistan . Preventive efforts are needed to start early in life and should continue throughout the life course . The most significant limitation of the present study is its small sample size of mi patients from punjab province, pakistan . Be that as it may, our findings could assist in future well - targeted intervention and awareness campaigns, aimed at residents of urban areas in particular, with a view to lessening the burden of chd.
Type 2 diabetes (t2d) is an increasingly widespread disease in both developed and developing countries . By the year 2030, it is predicted that more than half a billion people worldwide will be affected by this disease . Not only does t2d burden patients with numerous associated health complications such as cardiovascular disease, nephropathy, neuropathy, and retinopathy, but t2d is also a substantial strain on health care budgets . Diagnosis of t2d is typically determined by fasting blood glucose and the oral glucose tolerance test that examines an individual's ability to dispose of a glucose load . Glycated hemoglobin (hba1c), on the other hand, provides information on glucose management during the months preceding the initial testing . Despite these systematic measures, up to 60 percent of t2d cases are never diagnosed . The reasoning behind this misdiagnosis, or lack of diagnosis, is likely due to the insensitivity of these assays at predicting prediabetic and diabetic threshold values . Early diagnosis of t2d is extremely important, as early interventions might delay or even prevent full - blown disease [58]. T2d is rarely a static condition, but rather one that evolves and changes over time during the lifespan of the individual . Indeed, clinical risk factors appear to cluster in certain individuals more than others and are often independent of body mass index (bmi) [12, 13]. It is these high - risk individuals that are most likely to benefit from early and aggressive lifestyle interventions . Considerable variation is also evident in the response of individuals to treatment as well as their susceptibility to diabetes - related complications . This variability both in disease progression and treatment response emphasizes the need for additional tools for predicting disease progression and treatment success . The term metabolomics is similar to that of older technologies such as genomics (dealing with genes), transcriptomics (dealing with gene transcripts), and proteomics (dealing with proteins). The metabolome is comprised of small intermediary molecules and products of metabolism, including those associated with energy storage and utilization, precursors to proteins and carbohydrates, regulators of gene expression, and signalling molecules . Thus, the metabolome as the entirety of metabolites represents a real - time functional portrait of the cell or the organism . The metabolome is influenced by a plethora of factors, such as diet, lifestyle, medications, gender, and age . In this regard, metabolomics becomes a very powerful tool as it views the effects of pathological factors from vastly different origins in a single measurement . Specific methods employed in the study of metabolomics include nuclear magnetic resonance (nmr), gas chromatography mass spectrometry (gc - ms), liquid chromatography mass spectrometry (lc - ms), capillary - electrophoresis mass spectrometry (ce - ms), and high performance liquid chromatography (hplc). Nmr measures differences in the magnetic properties of atomic nuclei, mass spectrometry measures differences in the mass and electrical charge of the metabolites, and chromatography distinguishes metabolites by differences in adhesion properties . While hyphenated ms methods have the advantages of high sensitivity, small sample volumes, and relatively low costs, nmr has a greater range of simultaneously detectable molecular species, as well as simple sample preparation and excellent reproducibility . In all platforms, targeted profiling, quantitative values for a preselected subset of metabolites are calculated . For accurate results, internal standards must be used to calibrate sample concentrations by adding reference substances of known concentrations . In mass spectrometry, for example, a stable isotope - labeled variant of the metabolite of interest is spiked into the sample . For nmr, a single concentration reference substance is added; however obtained values may need to be scaled by metabolite - specific individual calibration factors [19, 20]. The results are a quantitative measurement of the abundance of the metabolites in the sample . For metabolic fingerprinting, all peaks of a spectrum, also known as features, are used for statistical analysis . After statistical analysis, the features of potential interest have to be assigned to their respective molecule using online metabolite databases such as the human metabolome database hmdb (http://www.hmdb.ca/) and the biological magnetic resonance database bmrb (http://www.bmrb.wisc.edu/). As researchers are constantly updating these databases with additional substances fingerprinting has the benefit of yielding a more comprehensive view of the metabolome and may help identify previously unknown disease mechanisms; however targeted approaches often have a lower variance and, thus, larger statistical power to detect small effects . As each platform (nmr, gc - ms, and lc - ms) provides unique information, with very little overlap in the detected metabolites, one way to circumvent this issue is to employ more than one system for a given study . Although this approach increases the number of detected metabolites and provides a more comprehensive picture, it is not typically performed due to increased costs and a lack of equipment and expertise . Samples most commonly analyzed via metabolomics include plasma, serum, and urine [22, 23], while other sample types such as cerebrospinal fluid and saliva are used less frequently . Metabolomics studies may also analyze tissue extracts; whole tissue by means of magic angle spinning (mas) nmr and even living organisms may be analyzed by in vivo nmr . There are no commonly used, standardized protocols for sample collection and storage for metabolomics studies, a fact that may contribute to additional variation in metabolomic profiles . For example, some studies collect samples only from fasting participants, while other studies do not have this as a prerequisite . Significant concentration differences between the metabolites of plasma and serum prepared from the same blood sample have been found . Although concentration differences have been found, plasma and serum metabolites correlate well with each other, indicating that both sample types allow accurate metabolomics measurements as long as serum and plasma samples are not compared to each other . While plasma showed higher over - time stability, serum allowed the quantification of more metabolites due to higher concentrations of selected metabolites . Storage conditions are a crucial factor for metabolomics studies, especially for prospective studies, where samples may be analyzed many years after being collected . Studies found no differences between plasma that was frozen immediately versus plasma that was stored at 4c for 8 and 24 hours before freezing, respectively . Likewise, no significant differences in metabolite profiles were found when comparing storage at 20c and 80c . Plasma samples stored at 80c for 13 to 17 years showed no influence of storage time on the metabolic profile . All these studies indicate that the choice of sample type (serum or plasma) and the storage conditions may be a minor issue for metabolomics studies . To examine metabolomic differences in response to t2d and pd in this review, we conducted a pubmed search for the following keywords: ((diabetes type 2) or (insulin resistance)) and (metabolomics or metabolomic or metabolite) and filtered for human studies published within the last 10 years . Only original articles were included, and studies that did not analyze serum or plasma were excluded . Only articles in english language were examined . These results were further filtered by including only papers dealing with diagnosis, prediction, prognosis, and translation of t2d . Most t2d metabolomics studies may be classified as either predictive or diagnostic . In this review, we define diagnosis as the identification of a currently occurring medical condition, while prediction is defined as the identification of subjects who will develop a certain medical condition in the future . Predictive studies follow initially healthy study participants for several years in prospective study designs . During follow - up such high demands reduce the amount of available study cohorts, and therefore most researchers have used samples from existing prospective studies including the framingham heart study (fhs) [34, 35], the cooperative health research in the region of augsburg (kora) study [36, 37], or the european prospective investigation into cancer and nutrition (epic) study [36, 37] for their research . Ideally, results from one study cohort are replicated in another, independent cohort, thus ensuring the validity of the results . Such studies have the advantage of testing at a single time point with no lengthy follow - up required . Studies with as little as 73 participants have led to reproducible results, while other studies of this type have used sample sizes of up to 7,098 individuals . An overview of the most commonly observed metabolite changes from predictive and diagnostic studies is shown in table 1 . Data on presymptopmatic individuals, those classified as having prediabetes (pd) or diagnosed as t2d, are reported . Pd, which includes impaired fasting glucose, impaired glucose tolerance, and insulin resistance, may be seen as an early form of t2d; diagnosis of pd often yields similar metabolomic results as t2d (table 1). In the following paragraphs, specific metabolites found to be predictive of t2d or pd will be discussed along with their physiological relevance . One group of metabolites consistently linked to pd and t2d in metabolomics studies is the branched - chain amino acids (bcaa) leucine, isoleucine, and valine . Elevated levels of bcaa have been found up to 13.7 years ahead of clinical manifestation [35, 36, 40], in pd [39, 4347] and in overt t2d [35, 37, 43, 46, 49, 50]. This association has led to the hypothesis of a causal role of these amino acids in the development of the disease . However, there is growing evidence that elevated bcaa levels may reflect a state of insulin resistance that is not necessarily specific to t2d as similar signatures have been observed for cardiovascular disease, chronic kidney disease, and ischemic stroke . Given this, the observed changes could also be indirect and primarily associated with insulin sensitivity rather than insulin secretion . In support of this hypothesis, bcaa have a plethora of biological functions, modulating protein synthesis and turnover as part of the mammalian target of rapamycin (mtor) pathway, facilitating glucose uptake in the liver and skeletal muscles, and also enhancing glycogen synthesis . Similarly, changes in aromatic amino acids (aa) have also been linked to the development of t2d years ahead of clinical manifestation [35, 36], pd [39, 44, 46, 47], and overt t2d [35, 46, 49]. Specifically, elevated circulating levels of the aa phenylalanine and tyrosine are linked to t2d . As for bcaa, a causative effect has yet to be fully elucidated, but changes in their circulating concentrations are thought to be indirect markers of insulin sensitivity . Several metabolomics studies have also found sugars, including glucose, mannose, fructose, and hexose, to be elevated 37 years before clinical manifestation of t2d [36, 41], in pd [37, 41, 43, 46] and overt t2d [37, 41, 43, 46, 49]. This discovery of glucose, which is one of the most standardized clinical parameters for t2d, amongst the top ranked predictive metabolites underscores its high importance for the risk assessment of t2d . Nonetheless, elevated sugar levels years ahead of overt t2d may also be caused by undiagnosed t2d amongst study participants . Additionally, metabolomics has shown an increase in -hydroxybutyric acid (ahba) up to 9.5 years ahead of t2d presentation [41, 42], in pd [41, 43, 4648] and t2d [41, 43, 46]. It is hypothesized that increased lipid oxidation, oxidative stress, and enhanced glutathione synthesis might explain the observed differences in ahba . Glycine, on the other hand, was found to be reduced 7 years before t2d [36, 37], as well as in pd [37, 38, 47] and in manifest t2d . This reduction in glycine is thought to be the result of increased gluconeogenesis as well as glutathione consumption driven by increased oxidative stress . Also, the abundance of incompletely oxidized fuels that are excreted as urinary acylglycine conjugates could reduce glycine levels . It has also been hypothesized that insulin deficiency leads to enhanced -aminolevulinate synthase 1 (alas - h) expression, in turn catalyzing the condensation of glycine and succinyl - coa into 5-aminolevulinic acid, thus reducing glycine levels . Several studies have also found changes in the ketones -hydroxybutyrate, acetone, and acetoacetate, which originate from fatty acid oxidation . Elevated levels were found 3 years ahead of disease manifestation as well as in overt t2d [41, 49]. For pd, a more complex picture is observed, where both elevated [41, 47] and reduced levels were found [39, 45]. These contradictory results are not surprising, as the role of fatty acid oxidation in the development of pd remains highly controversial . While some studies suggest that pd develops secondary to diminished fatty acid oxidation by cytosolic lipid accumulation, thereby impairing insulin signaling, other studies suggest pd to be characterized by excessive fatty acid oxidation, metabolic inflexibility, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle . These contradictory findings from different metabolomics studies highlight the complexity of the progression to t2d and the need for further research in this area . Glyoxylate levels were found to be increased 3 years ahead of disease onset, in pd and in overt t2d . Authors of this particular study hypothesized a connection to hypertension, as glyoxylate is a substrate of alanine - glyoxylate aminotransferase-2 (agt2), which regulates hypertension . A suppression of agt2 activity may be associated with the development of hypertension and increased glyoxylate . Likewise, 2-aminoadipic acid was reported to be elevated in individuals up to 12 years before t2d it is hypothesized that this metabolite is part of a compensatory mechanism upregulating pancreatic insulin secretion to maintain glucose homeostasis in early insulin resistance . To date, the latter two metabolites, 2-aminoadipicacid and glyoxylate, have only been identified as predictive by a single study, respectively . Further research on another t2d cohort is necessary to validate these results . Differences and predictive profiles for diacyl - phosphatidylcholine c32:1, elevated levels were detected years ahead of t2d and also seen in pd and in overt t2d . Although hyperlipidemia is common in t2d, decreased values were detected when analyzing single lipid species . Decreased levels of sphingomyelin c16:1 and acyl - alkyl - phosphatidylcholine c34:3 were found ahead of t2d and in overt t2d, with the latter being decreased also in pd . A lipid that was found in several predictive studies is lysophosphatidylcholine (lpc) c18:2, which was reduced up to 7 years ahead of t2d [3537], in pd and in incident t2d [35, 37]. Consistently, linoleoyl - glycerophosphocholine (lgpc) was found to be reduced 9.5 years ahead of t2d and in pd [47, 48]. As lgpc is one of the isomers of lpc c18:2, the association found in lpc c18:2 is likely due to the lgpc fraction contained in the total lpc c18:2 level . Lgpc has been shown to increase glucose - dependent insulin secretion in a cell line derived from beta cells, an effect that has been linked to the orphan g - protein - coupled receptor gpr119 . On the other hand, reduced lgpc levels have been linked to reduced cpla2 transcription, which would lead to an increased glucose uptake by adipocytes via a decrease in arachidonic acid . While the aforementioned studies all analyzed plasma or serum, there are a few reports employing other sample types . A study on urine found increased levels of glucose and glycine in overt t2d . In saliva, 1,5-anhydroglucitol has been found to be a marker for short - term glycemic control, something that has already been found in blood levels of this diet - derived metabolite . The value of these metabolomics based analyses in the context of t2d prediction and diagnosis has yet to be determined . The use of prediction measures can give an insight on the usefulness of certain metabolites in disease prediction . One of the most common means to assess prediction power is receiver operating characteristic (roc) curves, for which the area under the curve (auc) is used as prediction measure . The auc, also called c - statistics, is a value between 0 and 1, where values close to 0.5 indicate no predictive power (random classification) and values close to 1 indicate perfect prediction . The study by floegel et al . Found that metabolite levels alone could predict t2d slightly better than the diabetes risk score (drs) based on risk factors such as lifestyle, diet, and anthropometry with an auc 0.849 for metabolites versus 0.847 for drs . Adding metabolite profiles to risk prediction always enhanced the prediction, with an auc of 0.912 for a prediction using drs, fasting glucose, hba1c, and metabolites . Similarly, walford et al . Found that metabolite profiles scored slightly higher than a genetic risk score (grs), with aucs of 0.874 versus 0.861 . Wang et al . Used a clinical model including age, bmi, and fasting glucose and found a c - statistic of 0.801, which improved to 0.805 when metabolites were included in the model . When only high - risk subjects were included in the analysis, the c - statistic increased from 0.52 to 0.66, indicating that high - risk patients benefit more from metabolic prediction than low - risk patients . Padberg and coworkers found an auc of 0.82 when using only metabolites compared to an auc of 0.79 when using only fasting glucose levels to predict t2d . In a second cohort, the value improved from 0.83 to 0.86, respectively, for the prediction of pd . Wang et al . Used a prediction based on age, sex, fasting glucose, and bmi, resulting in a c - statistic of 0.91, which improved to 0.92 when including 2-aminoadipic acid . The net reclassification improvement (nri), an alternative measure for prediction power for these models, increased from 0.22 to 0.49 . In the study by wang - sattler and coworkers, the auc of a model containing age, sex, bmi, physical activity, alcohol intake, smoking, systolic bp, and hdl was 0.742 and increased to 0.754 when including metabolites . When including hba1c, fasting glucose, and fasting insulin into the model, the values were 0.818 and 0.828, respectively . . Found an auc of 0.762 when including familial diabetes, sex, age, and bmi . This value increased to 0.790 when including metabolites . In a second study, the values were 0.766 and 0.783, respectively . The objective interpretation of these numbers is hampered by the use of different combinations of clinical parameters to estimate t2d risk . While some studies use only fasting glucose, some use complex combinations of classical risk factors . This makes it impossible in some cases to determine whether metabolite signatures really improve t2d prediction when compared to the best available classical risk factors . Still, some studies use an extensive number of classical risk factors and obtain improved results when adding metabolite profiles to their prediction . This selective overview shows some of the drawbacks of metabolomics studies: the results often differ between studies and at times can even contradict each other . One reason for this may be that different analytical platforms measure different subsets of the metabolome, often with little overlap between different platforms . Other reasons include different characteristics of the observed populations (age and genetic background) as well as the use of different data analysis techniques . Granting all this, t2d risk prediction years ahead of overt disease has been shown to be feasible using metabolomics . As mentioned, a set of 12 metabolites or metabolite classes has been identified as predictive for t2d (table 1), a substantial number that could possibly be integrated into standard, bench top laboratory tests for diagnostic purposes . Apart from diagnosing or predicting the onset of pd or t2d, another important field for metabolomics research is prognosis, eluding disease course and outcome after its onset . The course and outcome of pd and t2d are to a large proportion driven by common complications including cardiomyopathy, nephropathy, peripheral neuropathy, and retinopathy . Unfortunately, in this area, little metabolomics research has been performed on human subjects, even though improved prognosis may enable early interventions, alleviate disease burden, and facilitate cost - effective treatments . It is estimated that 6070 percent of individuals with t2d have some form of cardiac dysfunction or cardiovascular disease . As a result, individuals with t2d are twice as likely as healthy controls to have heart disease or stroke, making cardiovascular disease (cvd) the number one complication of diabetes . Wu et al . Used metabolomics to investigate specific cardiovascular risk factors including high blood pressure, nonalcoholic fatty liver disease, and coronary heart disease in a group of t2d patients . Unfortunately, this study only analyzed metabolic differences between these three risk factors and combinations thereof and did not include a control group without cvd risk factors . Although the study could show notable metabolic differences between these diseases and their combinations, the authors state that the value of the results is limited as they were not able to recruit a matching control group without t2d complications . Likewise, another study failed to predict coronary artery disease in 190 t2d patients within 4 years of follow - up . For diabetic nephropathy, sharma et al . Compared urine metabolites of diabetes patients with and without chronic kidney disease . Although both type 1 and type 2 diabetes patients were enrolled in the study, only t2d cases were used in the screening cohort . It was found that 13 urinary metabolites were significantly reduced in chronic kidney disease patients . Interestingly, 12 out of these 13 metabolites were found to be connected to mitochondria, and as such the authors suggested impaired mitochondrial metabolism in diabetic kidney disease . However, reduced urinary levels may also stem from reduced kidney function and thus not impaired mitochondrial function per se . In a rare prospective study, niewczas and colleagues analyzed the plasma of 40 t2d patients who developed end - stage renal disease within 812 years of follow - up and compared it to a matched control group . Uremic solutes and acylcarnitines were significantly increased prior to end - stage renal disease, hinting at early reductions in renal function that did not show up in standard renal function tests based on glomerular filtration rate estimated from serum creatinine levels . A set of aa and their derivatives showed decreased levels; notably, bcaa and aromatic aa as well as ahba were significantly decreased, suggesting enhanced mitochondrial aa -oxidation . Of interest, these metabolites were decreased rather than increased as usually seen for both the prediction and diagnosis of t2d (table 1). The results of this study have not yet been replicated in an independent cohort, but another study comparing t2d with and without diabetic nephropathy also found reduced levels of bcaa and aromatic aa . Finally, a study has been conducted on diabetic retinopathy, which is the leading cause of blindness and visual impairment among working - age persons in developed countries, finding reduced levels of galactitol and ascorbic acid in vitreous humor . However, vitreous humor is a hard - to - collect sample type, which limits the application of this kind of analysis . Additionally, the control group in this study was not made up of healthy individuals but of patients with a macular hole . All in all, the study of t2d - associated complications is still underrepresented in field metabolomics research, especially regarding prospective studies on human samples . Further investigations similar to that of niewczas et al ., who prospectively analyzed metabolomic changes ahead of end - stage renal disease in t2d patients, have the potential to precipitate early interventions based on individual risk assessments . Translation into clinical practice is defined as enabling the use of results from basic metabolomics research to enhance diagnosis, prediction, prognosis, and therapy . In the context of metabolomics, this is achieved by defining a predictive or diagnostic profile for a specific population (age and sex) under a defined set of conditions (fed and fasted). Basic research metabolomics studies typically measure hundreds of metabolites, an approach that is not feasible or cost - effective for large - scale application . Additionally, laboratory equipment used for metabolomics studies is expensive and not readily available at the point of care . For these reasons, only a small subset of relevant metabolites that can be assessed using standard equipment or assays could be incorporated into a clinical routine . Although much more work needs to be done, such measures have the potential to be quick, cost - effective, and relatively easy to interpret . Identification of a high probability of t2d risk in a presymptomatic patient could inform physicians of the need for early testing, intensive intervention, and continued monitoring . This approach has the potential to be far more effective compared to treating full - blown t2d, where irreversible damage may have already occurred . Thus, the translation of metabolomics research into clinical application may lead to informed decision - making in the realm of personalized medicine . To date, work translating metabolomics findings into clinical application has been limited, and most studies have focused on t2d prediction rather than t2d comorbidities . There may also be value in combining metabolomics data with other molecular and clinical datasets . A good example of this integration can be found in walford et al ., who analyzed a combination of 62 genetic and 9 metabolic biomarkers for the purpose of predicting t2d risk . Results showed that biomarkers such as the bcaa isoleucine, the aromatic aa phenylalanine and tyrosine, triacylglycerides, phosphatidylcholines, and lysophosphatidylcholines were found to be predictive up to 13.4 years before the onset of disease . When metabolomic, genotypic, and clinical data were analyzed together there was a significant rise in predictive performance, suggesting that these datasets can be used to compliment or even validate each other . Of note, the authors of this study state that they may not have used the most ideal combination of biomarkers . Investigators varvel and colleagues have also made significant advancements in translating metabolomics research into clinical application . Specifically, they assessed a panel of 19 blood - based biomarkers, including metabolites such as glucose and ahba, proteins, and antibodies . Based on these values, they analyzed t2d risk in 1,687 patients presenting for risk assessment . Patients belonged to an obese high - risk cohort, with 48 percent of patients meeting the criteria for metabolic syndrome and one - third having been previously diagnosed with t2d and/or hypertension . Screening by classical clinical parameters classified 929 patients (55.1%) as normoglycemic . When classifying using biomarker analysis, 766 out of these normoglycemic patients (82.5%) were classified as at - risk . This indicates that a large amount of high - risk patients is missed when relying solely on classical clinical parameters . The classification results were presented to the patient and their physician and included treatment considerations, with the final treatment decision made exclusively by the treating physician . During care, guided by biomarker testing, a significant amount of patients initially classified as although these results are promising and represent an example of how informed decision - making can be based on metabolomics data, a randomized study design will have to be employed in order to authenticate these results . It is clear that further research on translating metabolomics results into clinical applications is necessary . In order for this to be successful, a robust set of relevant biomarkers must be defined, considering also that the metabolites have to be measured cost - effectively in a standard clinical environment . In any case, it may be feasible for metabolomic biomarkers to be used in addition to established clinical tests for t2d and its comorbidities . Day - to - day variations in the metabolomic profile, which occur due to differences in diet, physical activity, and so on, may be minimized when analyzing serial time points of the same individual to create an average metabolic profile . Additionally, insights from metabolomics studies may be the basis for genome - wide association studies (gwas) [7375], possibly identifying genetic backgrounds of the observed effects and further strengthening the personalized aspects of diagnosis and treatment . The field of metabolomics is still relatively young and exists at a basic research level, where the issues of measurements and data analysis often require novel solutions . Moreover, metabolomics data often lacks standardization in its reporting, thus making it difficult to compare results from independent studies . In addition, differences in protocols and instrumentation can yield varied sets of detectable metabolites, often with little overlap to other designs . Even when similar methods are used, different statistical approaches might lead to contradictory results . Despite these potential limitations, metabolomic studies have the potential to determine a unique set of metabolites that are predictive of both pd and t2d, often years or decades ahead of disease onset . This powerful information may shed light on disease development and may improve patients' health, as shown in the pilot study by varvel et al ., who used a set of metabolites in a clinical setting to inform physicians on t2d risk factors . Although there is much work left to do, the evidence of metabolomics benefitting t2d care makes its clinical application inevitable.
Endovascular occlusion with detachable platinum coils has become an established treatment for managing patients who have ruptured intracranial aneurysms (1 - 3). Although numerous studies regarding endovascular or surgical treatment for ruptured intracranial aneurysms have been reported in the literature, there are only two randomized, prospective studies comparing endovascular coiling and surgical clipping to date (4 - 6). The results of the first, prospective randomized trial from finland compared endovascular and surgical treatment of ruptured intracranial aneurysms was published in 1999 and 2000 (4, 5). The study included 109 patients with acute (<72 hours) aneurysmal subarachnoid hemorrhage who were suitable for both endovascular coiling and surgical clipping . Aneurysms were considered suitable for both coiling and clipping if: 1) the neck of the aneurysm was smaller than the fundus; 2) it was not a fusiform aneurysm; 3) the neck of the aneurysm and relationship to the parent vessel was distinguishable; and 4) the diameter of the aneurysm was bigger than the smallest coil, or 2 mm, resulting in approximately half of the patients with aneurysmal subarachnoid hemorrhage suitable for randomization . As an immediate postembolization angiographic result, posterior circulation aneurysms showed more complete occlusion with coiling than with surgery (p = 0.045), whereas anterior circulation (anterior cerebral artery, anterior communicating aneurysm, and pericallosal artery) aneurysms showed the better angiographic outcome with surgical clipping than with endovascular coiling (p = 0.005). The procedure - related mortality rate was 2% in the endovascular group and 4% in the surgical group . At 12 months, the clinical outcome did not significantly differ between two groups, as 79% of endovascular versus 75% of surgical patients had good or moderate recovery on glasgow outcome scale (p = 0.3). As 12-month angiographic results, 76.9% of the endovascular group showed total obliteration of the aneurysm, whereas 86.0% of the surgical group showed total obliteration, which was not statistically significant . The authors concluded that the clinical outcome at 3 and 12 months was comparable after endovascular and surgical treatment of ruptured intracranial aneurysms . Isat was a randomized, multicenter trial compared surgical clipping with endovascular coiling in patients with ruptured intracranial aneurysms (6 - 8). Between 1994 and 2002, 2143 patients were enrolled in the study from 43 neurosurgical centers, most in the united kingdom and europe . The primary objective of the isat was to determine whether there was a reduction in the proportion of patients who were dead or dependent (defined as modified rankin scale, mrs 3 - 6) at 1 year by following a coiling strategy compared with a clipping strategy . Recruitment of patients was stopped after an interim analysis showed a benefit of endovascular treatment on the primary outcome at 1 year . The complete 1-year data from isat was published in the lancet in 2005 (7). Two - hundred - fifty of 1,063 (23.5%) patients allocated to endovascular coiling were dead or dependent at 1 year, compared with 326 of 1,055 (30.9%) patients allocated to surgical clipping: an absolute risk reduction of 7.4% (95% ci 3.6% to 11.2%, p = 0.0019). The early survival advantage in the coiled group was maintained for up to 7 years and was statistically significant (log rank p = 0.03).the risk of epilepsy was substantially lower with endovascular coiling than with clipping: relative risk 0.52 (95% ci 0.37 to 0.74). There was no significant difference in the frequency of rebleeding between the groups (log rank p = 0.22). The rebleeding risk after 1 year in the coiled group is approximately 0.2% per patient year with follow - up from 1 to 8 years with a mean of 4 years . Isat concluded that endovascular coiling of ruptured intracranial aneurysms, when a patient is in good clinical grade and the aneurysm anatomy is suitable for endovascular treatment, is more likely than neurosurgical treatment to lead to independent survival at l year . Since the 1-year results of isat study were released, treatment of patients with a ruptured intracranial aneurysm has changed significantly over the past decade . In many centers, coiling has become the mainstay of treatment of ruptured intracranial aneurysms when both coiling and clipping are considered suitable . The long - term follow - up results of isat were published in the lancet neurology in 2009 (8). At 5 years six - hundred - twenty - six of 755 (83%) patients treated by endovascular coiling were independent at 1 year, compared with 584 of 713 (82%) patients treated by surgical clipping . However, the risk of death was still significantly lower in the coiling group than in the clipping group (relative risk 0.77, 95% ci 0.61 - 0.98; p = 0.03). At 5 years, 112 of 1,046 (11%) patients in the coiling group have died, compared with 144 of 1,041 (14%) patients in the clipping group (log rank, p = 0.03). The annual risk of the treated aneurysm rebleeding is higher in the patients treated by the coiling than in those treated by the clipping: 10 rebleeding occurred in the coiling group and 3 in the clipping group . However, the risk remains low and is at a similar level to the risk of further subarachnoid hemorrhage (sah) from another source, either a pre - existing aneurysm or a newly formed aneurysm . The main finding of the study is that rates of independence become essentially equivalent at 5 years, whereas mortality rates still favor the patients treated by endovascular coiling . The major contribution to the achievement of equivalent long - term functional outcomes might be the continuing improvement in the neurological function of less severely disabled patients in the clipping group between year 2 and year 5 ., isat has demonstrated that endovascular coiling of ruptured intracranial aneurysms has benefits over surgical clipping in those patients suitable for either treatment, but this difference decreases over time (9). With the significant advances in the imaging techniques and widespread use of noninvasive intracranial imaging studies such as ct or mr angiography, the incidental detection of unruptured intracranial aneurysm (uia) has increased greatly . The management of patients with uias becomes an important public health issue, but still remains a controversial topic . There has been no published randomized, controlled trial with regard to compare natural history and coiling or clipping, or clipping and coiling for the management of patients with uias, but one such trial is currently ongoing . International study of unruptured intracranial aneurysms (isuia) is the largest natural history study of uia (10,11). A prospective cohort study from isuia showed that the 5-year cumulative rupture risk of uias smaller than 7 mm in the anterior circulation with no history of sah to be 0% and that with a history of sah 1.5% (11). Aneurysms at posterior circulations (basilar tip and the posterior communicating artery), aneurysms larger than 10 mm, and aneurysms that are found in patients who had bled from a prior aneurysm were found to have higher risks (about 0.5% per year). Table 2 shows the 5-year cumulative rupture rates of uia among patients in the unopearated prospective cohort of isuia . Isuia data provided the first, international prospective data set, but there have been many criticisms of the isuia study, especially with regard to patient selection bias . Detailed discussion about these criticisms is out of range for this review and is well described elsewhere . It is generally believed that the risk of rupture for a uia is approximately 1% per year for lesions 7 - 10 mm in diameter observational study published in 2009, ishibashi et al . Reported that the annual rupture rate for 529 uias was 1.4% during mean follow - up period of 905.3 days (14). In their study, a previous history of sah (the hazard ratio 7.3; 95% ci, 2.5 to 21.2), posterior circulation aneurysm (the hazard ratio 2.9; 95% ci, 1.1 to 8), and large size were significant independent predictors for aneurysm rupture . The hazard ratio in the large sized (> 10 mm) and giant (> 25 mm) aneurysm were 12.3 and 50 compared with that for small sized (<5 mm) uias . More recently, a prospective multi - center, observational study to determine the optimal management for small uias has been published by japanese group (15). In that study, 448 uias <5 mm in size the annual rupture rate for small uias smaller than 5 mm is quite low: 0.54% overall, 0.34% for single aneurysms, and 0.95% for multiple aneurysms . Patient <50 years of age, aneurysm diameter of> 4 mm, hypertension, and aneurysm multiplicity were found to be significant predictive factors for rupture of small uias . Other reported risk factors for rupture of uias in the literature include cigarette smoking and female sex . A recently published systematic review and meta - analysis found an overall risk of poor outcomes (mrs 3 - 6) is in the range of 4 - 5% after endovascular coiling in patients with uia (16). Recurrence rate is high: 24.4% of the patients followed up for 0.4 - 3.2 years . The annual risk of bleeding per patient - year after endovascular coiling was 0.2%, but the lack of systematic follow - up, short observation periods, and missing data hampered the assessment of clinical efficacy of endovascular coiling (16). A rct is ongoing to answer the question whether active treatment can improve the outcome of patients with uia as compared with observation . Team trial (trial on endovascular aneurysm management) is an international, randomized, multicenter, controlled trial comparing endovascular treatment versus conservative management of uias (17). Primary endpoint is mortality and morbidity (mrs 3 - 6) from intracranial hemorrhage or treatment . Secondary endpoints include incidence of hemorrhagic events, morbidity related to endovascular coiling, morphological results, overall clinical outcome and quality of life . The study will be conducted in 60 international centers and will enroll 2,002 patients equally divided between the two groups . The duration of the study is 14 years, the first three years being for patient recruitment plus a minimum of 10 years of follow - up . Preventive treatment with coiling or clipping in patients with uia can be justified when the benefits outweigh the risks of such treatments, with strong evidence supported by valid data . Currently, in the absence of randomized controlled studies, any proposed treatment algorithms for uias remain invalid (16, 17). In conclusions, short - term results of isat study showed that endovascular coiling of ruptured intracranial aneurysms, when a patient is in good clinical grade and the aneurysm anatomy is suitable for endovascular treatment, is more likely than neurosurgical treatment to lead to independent survival at l year . Long - term follow - up results of isat study showed that rates of independence become essentially equivalent at 5 years, whereas mortality rates still favor the patients treated by endovascular coiling . Thus, isat study has demonstrated that endovascular coiling of ruptured intracranial aneurysms has benefits over surgical clipping in those patients suitable for either treatment, but this difference decreases over time . Because rct comparing conservative management with surgical clipping and with endovascular coiling have not been performed to date for unruptured intracranial aneurysms, a rct is ongoing to answer the question whether active treatment can improve the outcome of patients with uia as compared with observation.
Trefoil factors (tffs) represent a family of small peptides (6 - 10 kda) characterized by a common sequence, consisting of 42 - 43 amino - acids and highly conserved in mammals 1 . Intramolecular disulphide bonds within the sequence cause the formation of the characteristic three - loop structure, called the trefoil domain 1, 2, making tffs stable molecules that are relatively resistant to proteases . Three mammalian trefoil peptides are known: tff1 (formerly ps2) and tff3 (intestinal trefoil peptide) contain one while tff2 (spasmolytic polypeptide) contains two trefoil domains 3 . Trefoil factor 1 is a small cysteine - rich acidic peptide consisting of 60 amino acids 1 . In normal human tissues, tff1 is predominantly expressed in the gastrointestinal tract 4 and, although its biological function is not clarified, it seems to have an important role in preserving the mucosa of the gastrointestinal tract 5 . In malignant tissue, tff1 was initially found in breast cancer 6, but it has also been detected in several other carcinomas, such as stomach, pancreas, large intestine, endometrium, ovary, uterus, bladder, and prostate 7 - 10 . Hormones (estradiol 11), growth factors such as insulin - like growth factor i (igf - i), igf - ii, epidermal growth factor (egf) or transforming growth factor (tgf) 11 - 13, phorbol esters 11, 14, plasminogen activator and fos and jun oncoproteins 11 have been reported to regulate the expression of the tff1 through the activation of the gene promoter containing an estrogen - response element (ere) and a tpa - response element (tre) 15 . The peptide was found to be overexpressed in approximately 50% of primary breast carcinomas 16, mainly estrogen receptor alpha - positive (er+) ones . It has been demonstrated that estrogen stimulation of an estrogen - dependent breast cancer cell line induce significant (up to 100-fold) increase of tff1 mrna as well as an increase of protein level 6 . Despite numerous studies, considering that low levels of tff1 were shown to be characteristic for normal breast tissue, it has been proposed that overexpression of the peptide in breast cancer indicates its adverse function, possible as an oncogene . Early studies indicated that tff1 might be a mitogen but they failed to demonstrate such biological function . Recent studies provided evidence regarding the possible role of tff1 in breast cancer contributing to tumor aggressiveness . It has been reported that tff1, induced by estrogen, stimulates migration of breast cancer cells 18, 19 . Moreover, tff1 dimer was shown to be more effective than tff1 monomer in stimulation of breast cancer cell motility 19, possible due to its interaction with a putative cell surface receptor that has not been yet identified . A role of an estrogen - regulated, autocrine motogenic factor was assumed to be a major biological role of tff1 in breast cancer . The finding that tff1 overexpression has been detected in approximately 50% of breast carcinomas 7, while only low levels of the peptide have been found in normal breast tissue 16 provided a rationale for numerous studies evaluating the potential clinical significance of tff1 in breast cancer . The protein is not only regarded as an indicator of the intact er signaling pathway 20, 21, it is considered to be a predictive factor for positive response to endocrine therapy in breast cancer patients 21 - 26 . Furthermore, some studies suggested that tff1 expression might be useful in identifying the subgroup of er - positive breast cancer patients being more responsive to aromatase inhibitors (ai) than to tamoxifen 27 . Most studies reported on association between tff1 expression in breast cancer and a favorable prognosis 16, 21, 25, 28 - 32 while some studies failed to confirm such correlation 33 . In our study, a significant difference in tff1 levels was found in relation to either menopausal status, histological grade, estrogen receptor (er) status or progesterone receptor (pr) status . The first aim of our study was to determine a biologically - related cut - off value for tff1 expression that would allow us to distinguish between estrogen - dependent and estrogen - independent tff1 expression in breast cancer . On the basis of tff1 level distribution in both premenopausal and postmenopausal patients bearing er - positive and er - negative tumors (regardless of histological grade), a concentration of 14 ng / mg of tff1 was established as a cut - off value for tff1 expression in breast cancer . The second aim of our study was to evaluate a potential clinical relevance of tff1 in breast cancer, on the basis of the established cut - off value . Therefore, we analyzed disease free - interval (dfi) probabilities in relation to tff1 status . Tff1 status allowed us to discriminate between patients with high versus low risk for development of distant metastases during a 3-year follow - up after the surgery . This retrospective study included tumor samples from 226 consecutive female patients with clinical stage i or stage ii breast cancer . After the surgery, primary operable invasive early breast carcinomas were histologically verified for all patients in the study . In the adjuvant setting of these patients, treated at the institute for oncology and radiology of serbia between 2002 and 2005, chemotherapy was given to 64 patients (32%), the same number of patients received hormonotherapy, both types of systemic therapy were administered to 32 patients (16%) while 41 patients (20%) received no adjuvant therapy . Histological specimens were reviewed and then classified according to the criteria of the international union against cancer for tn stages 34 and according to the criteria for histological type 35 and grade 36 . Patients were considered to be postmenopausal if menstruation ceased for at least six months, otherwise they were considered as premenopausal . Patients' follow - up was conducted every three months for the first two years and every six months during the third year . The recurrence of the disease was set as an end - point in our study because it is clinically useful in regard to metastatic disease progression . Disease - free interval was defined as time from the initial diagnosis to the emergence of distant metastases . The follow - up data were available for 201 patients and these patients were included in the survival analysis . Forty - three (21.4%) patients developed distant metastases during a 3-year follow - up . The study was approved by the ethics committee of the institute of oncology and radiology of serbia and the patients provided their informed consent before entering the study . Estrogen receptor and progesterone receptor levels in cytosol extracts of tumor samples were measured by using the classical biochemical method as recommended by the european organisation for research and treatment of cancer (eortc) 37 . The intra - laboratory quality assessment of the steroid hormone receptor levels was performed periodically following the eortc recommendation 38 . The cut - off value for the quantitative classification of positive receptor status was 10 fmol / mg for er and 20 fmol / mg for pr 39 . Tff1 levels were assayed in tumor cytosols obtained for routine er and pr determination and were measured by using a solid - phase radioimmunoassay (elsa - ps2 cis biointernational, gif - sur - yvet, france). The distribution of tff1 levels between the subgroups of patients was assessed using mann - whitney or kruskal - wallis test . The distribution of different therapies between subgroups of patients was evaluated by chi - squared test . The distribution of tff1 levels within the analyzed group of 226 breast carcinomas was statistically different from the normal distribution (kolmogorov - smirnov test, p<0.001) and it is presented in figure 1 . A wide range of tff1 levels was detected in breast carcinomas and protein levels varied from 1.5 to 135.8 ng / mg protein, with a median value of 9 ng / mg protein . The distribution of tff1 levels in relation to tumor - host (age, menopausal status) and tumor parameters (axillary node status, tumor size, histological grade, histological type, er and pr status) is shown in table 1 . Statistical analysis revealed that tff1 levels were significantly higher in premenopausal than in postmenopausal patients (p=0.02) as well as in patients bearing histological grade i or ii tumors compared to those bearing histological grade iii tumors (p<0.001). In addition, er - positive (er+) or pr - positive (pr+) tumors were characterized with higher tff1 levels in comparison to er - negative (er-) or pr - negative (pr-) tumors, respectively (p<0.001 in both cases). The last result may be expected, considering a statistically significant correlation between tff1 and either er or pr levels in analyzed samples of breast carcinoma (r=0.295, p<0.001 and r=0.254, p<0.001, respectively). Regardless of significant differences in tff1 levels between the indicated groups of patients, the ranges of peptide levels between corresponding groups were comparable (as shown in table 1) and we were unable, on the basis of these results, to define biologically - related cut - off value for tff1 expression in breast carcinomas . In an effort to discriminate estrogen - independent from estrogen - dependent tff1 expression, the distribution of tff1 levels was, further, analyzed between the groups of patients defined by combining menopausal status, histological grade, er status and pr status . Figure 2 shows the distribution of tff1 levels in er - positive and er - negative postmenopausal patients bearing tumors of different histological grade . Among postmenopausal patients, those with er - negative status had significantly lower tff1 levels in comparison to those with er - positive status, irrespective of tumor histological grade (p=0.04, p<0.001 and p<0.05 for histological grade i, the ranges of tff1 levels of postmenopausal patients with tumors of different er status (er+ vs. er-) were not comparable within the same histological grade of tumor . Since the highest tff1 levels in er - negative tumors of postmenopausal patients were similar across tumors of different histological grade (12.3, 13.9 and 9.6 for histological grades i, ii and iii, respectively), we established 14 ng / mg as the cut - off value to discriminate between estrogen - dependent and estrogen - independent tff1 expression in breast cancer . Accordingly, we were able to define tff1 status as a positive (tff1 +) or a negative one (tff1-), depending on whether tff1 levels in tumors were higher or lower than the cut - off value . Statistical analysis showed that tff1 status alone did not show a significant association with dfi probabilities during the first 3 years of patients' follow - up (data not shown). Therefore, we compared dfi probabilities between groups of patients defined by different clinico - pathological parameters, focusing to parameters that appeared to affect tff1 level distribution in breast cancer . Analysis of premenopausal patients bearing er - positive and pr - positive tumors of histological grade ii revealed a statistically significant difference in dfi probabilities between patients with positive- and negative tff1 status, where those with positive tff1 status had significantly lower dfi probabilities (p<0.05, figure 3). In addition, postmenopausal and pr status were found to affect dfi probabilities of tff1-positive patients with er - positive tumors of grade ii in such a way that: a) among patients with positive pr status, premenopausal patients had significantly lower dfi probabilities in comparison to postmenopausal ones (p=0.009, figure 4) and b) among postmenopausal patients, those with negative pr status had significantly lower dfi probabilities in comparison to those with positive pr status (p=0.04, figure 5). By use of chi - squared test, analysis revealed no difference in distribution of adjuvant systemic therapies among analyzed subgroups of patients during the first 3 years of follow - up (data not shown). The distribution of tff1 levels in our study (figure 1), in addition to a wide range of protein levels, implies that breast cancer is a heterogeneous disease . Our finding that high tff1 levels were related to premenopausal patients is in accordance with the findings of other studies 21, 41, although some opposite findings were also reported 32 . Considering that middle - aged group (45 - 59 years old) consisted of both premenopausal and postmenopausal patients while all patients younger than 45 were premenopausal and all patients older than 59 were postmenopausal in our study, age of the patients was not analyzed as a continuous variable, but stratified into three age subgroups . Although such stratification might be a possible reason why a significant difference in tff1 levels was not found in relation to age of patients in our study, our results confirm some literature data showing that expression of some er - inducible proteins, including tff1, are not significantly related with the age at diagnosis 42, 43 . Our results also confirm the results of some previous studies reporting that tff1 expression is negatively associated with histological grade of tumor 21, 30, 32, 44 while positively associated with er 21, 29, 45 or pr positivity 21, 30, 46 . Considering that a negative correlation between histological grade and er positivity of tumors has been demonstrated previously 47, results of our study are not surprising . However, not all the studies have demonstrated such association between tff1 expression and er 48 or pr positivity 49 or histological grade 29, 32 of tumor . The majority of studies, including ours, failed to demonstrate any association between tff1 and lymph node involvement or tumor size 21, 30, 32, 50 which may indicate that tff1 expression is not related to the stage of tumor development . However, a negative association between tff1 expression and lymph node involvement 27, 44 or tumor size 44 has been reported in some studies . The choice of an optimal tff1 cut - off value may be essential for assessing a potential clinical relevance of tff1 in breast cancer as it may lead to a better stratification of breast cancer patients' subgroups . The result of such stratification should contribute to the improvement of patients' treatment in a way to apply treatment at an earlier stage of the disease or to avoid unnecessary risk for patients who will not benefit of an additional treatment . In many studies, various statistically - related values were used as cut - off values in order to discriminate between tff1-positive and tff1-negative patients that might be at different (low / high) risk for developing distant metastases after primary therapy . In our study, we applied a different approach aiming to find a biologically - related cut - off value that would allow us to discriminate between estrogen - dependent and estrogen - independent tff1 expression in breast carcinoma . As we were unable to determine such a cut - off value just on the basis of tff1 level distribution in relation to either menopausal status, histological grade, er status or pr status, we performed additional analysis of the distribution of tff1 levels in relation to er and/or pr status among patients bearing tumors of different histological grade . The additional analysis was performed both within the group of postmenopausal (figure 2) and within the group of premenopausal patients (data not shown). Within the group of postmenopausal patients, the analyses of tff1 levels revealed that: er - negative patients had significantly lower protein levels in comparison to er - positive ones, regardless of histological grade of tumor, the range of tff1 levels for er - negative patients was not comparable with the one corresponding to er - positive patients, regardless of histological grade of tumor, the highest tff1 level of er - negative patients was less than 14 ng / mg protein . Er - negative patients had significantly lower protein levels in comparison to er - positive ones, regardless of histological grade of tumor, the range of tff1 levels for er - negative patients was not comparable with the one corresponding to er - positive patients, regardless of histological grade of tumor, the highest tff1 level of er - negative patients was less than 14 ng / mg protein . Among premenopausal patients, however, tff1 levels in er - negative tumors were neither significantly different from the corresponding levels in er - positive tumors nor lower than 14 ng / mg, within all three histological grades . It is well known that postmenopausal women have significantly lower estrogen levels in comparison to premenopausal ones . Nevertheless, these low estrogen levels in postmenopausal women are still able to induce estrogenic response in a way to stimulate tff1 expression in breast cancer cells . In addition, tff1 expression in breast cancer may also be induced by growth factors, such as igf - i 15, 25, via signaling pathway that are related or not to er, thus making it estrogen - independent . On the basis of our results, we assumed that tff1 expression is predominantly regulated by growth factors in er - negative tumors of postmenopausal patients while additionally regulated through indirect estrogen - dependent er signaling pathways in er - positive tumors of these patients 51 . We further assumed that indirect estrogen - dependent er signaling pathways may dominantly regulate tff1 transcription in er - negative tumors of premenopausal patients while a classical, estrogen - dependent er signaling pathway is supposed to be dominant in er - positive tumors of these patients . Based on these assumptions, the highest tff1 level in er - negative tumors of postmenopausal patients was considered as a maximum of estrogen - independent tff1 expression . A value of 14 ng / mg of protein was established as the cut - off value to distinguish between estrogen - dependent and estrogen - independent tff1 expression . Accordingly, patients with tff1 levels lower or higher than the cut - off value were regarded as those with tff1-positive or tff1-negative status, respectively . It should be noted that pr levels were significantly correlated to tff1 levels in our study but they seem to have no effect on estrogen-(in)dependent tff1 expression in breast carcinoma . It may be assumed that pr and tff1 participate in er signaling in breast cancer not through common but distinct pathways . Some differences in clinico - pathological and biological characteristics between pr and tff1 have already been considered by other investigators hypothesizing that estrogen - independent signaling of tff1 is different from the one of pr in post - menopausal women with er - positive breast cancer 27 . A question arises on whether a similar difference in tff1 and pr signaling pathways in breast cancer may be hypothesized in case of estrogen - dependent signaling of tff1 and we believe it may be . Detection of low tff1 expression in normal breast tissue and its high expression in majority of breast carcinomas has been the rationale for numerous studies on the potential clinical value of tff1 expression in breast cancer 52 . Despite the increasing amount of data on this subject, the findings are inconclusive . In many studies, especially in those using cytosol - based assay, numerous studies also suggested that tff1 expression may be a predictive indicator of positive response to endocrine therapy in er - positive breast cancer patients and, moreover, an indicator of sensitivity to ai over tamoxifen . Tff1 expression has even been identified as an informative marker for the detection of micrometastases 53 . In several studies, however, the prognostic or predictive value of tff1 expression in breast cancer has not been demonstrated 27,30 . Studies on the clinical value of tff1 in breast cancer may differ among each other in many issues . Cohorts of patients, the length and end - points of follow - up, methodologies used for determination of tff1 expression, cut - off values for tff1 positivity, etc . Represent some of the variables that may affect the findings as well as the conclusions of such studies . The follow - up after the surgical therapy is not usually a predefined period of time and differences in patients' follow - up exist within as well as among studies, even when the same end - points are considered . In order to avoid the possible adverse impact of the varying follow - up, a requirement of an equal follow - up period (3 years) has been set up for all patients included in the study . It has previously been reported that a clinical relevance of a biomarker may change in time following the surgical treatment . Several studies reported on the potential of er status as a prognostic indicator in the early follow - up (mostly 2 - 2.5 years after the surgery), as its prognostic strength weakened over time 54, 55, while others reported on prognostic relevance of er status in the late follow - up 56 . A time - varying effect of nodal status, tumor size and histological grade on prognosis of breast cancer patients has also been reported by several studies 54, 57 . Considering potentially time - dependent clinical relevance of a biomarker, the choice of an optimal follow - up seems important in order to obtain a relevant evaluation of an indicator of the clinical outcome . The emergence of distant metastases in breast cancer has been shown to follow a double - peak pattern with an early peak at about 2 - 3 years after surgery and a late peak at about 5 years after surgery 55, 58 . It has been proposed that the surgical therapy induce an increase of probability for the transition of non - dividing single cells to a state of avascular micrometastases 59 and, further, to a phase of vascularization and further growth 60, resulting eventually in an early peak . It is well known that, in terms of breast cancer - specific overall survival, the shorter the interval, the shorter the patient is expected to survive . Given the importance of the early follow - up and by considering a possible time - dependent clinical relevance of a biomarker, it seemed reasonable to us to assess the potential clinical value of tff1 in primary operable breast carcinoma during the first 3 years of follow - up . Such investigation was also based on our finding that no difference in dfi probabilities was found between groups of patients who were subjected to different modalities of adjuvant systemic therapy: no therapy vs. chemotherapy vs. hormonotherapy vs. chemo+hormonotherapy (data not shown). Our principal aim was to eventually identify subgroups of primary operable breast carcinoma where tff1 expression might help to discriminate between patients at low vs. high risk for development of distant metastases in a 3-year follow - up after the surgery . Some previous studies have suggested that high expression of tff1 is associated with breast carcinomas of a more benign course 27, 32, 44 . Our results are, partially, in contrast with such finding and suggest a possible adverse effect of high levels of tff1 in breast carcinoma (at least in some of its specific subgroups). It has been presumed that tff1 might act as a mitogen but early studies failed to demonstrate such biological function . Numerous studies have reported on a role of tff1 in stimulating the motility and invasion of breast cancer cells 17, 19, 21 . By acting as a motogen, tff1 was suggested to promote cell dissemination and development of metastases in breast cancer, two processes associated with more aggressive tumor behavior . A positive role of tff1 in breast cancer cell migration might provide a rationale for our result, showing a worse outcome for tff1-positive premenopausal patients bearing er - positive and pr - positive tumors of histological grade ii, in comparison to these patients with negative tff1 status (figure 3). As previously mentioned, it may be assumed that tff1 exerts its function through a classical er signaling pathway that might be predominant in tumors of premenopausal patients due to high levels of estrogen . Additional reason for a worse outcome of these tff1-positive patients may be a potential role of tff1 in proliferation of breast cancer cells since increased levels of tff1 have been demonstrated to stimulate this process in breast cancer 17 . A potential role of tff1 in proliferation and/or migration of breast cancer cells might be further indicated by the result showing that none of these tff1-negative patients developed distant metastases during a 3-year follow - up . This result indicates that tff1 might significantly affect migration and/or proliferation when its concentration is high enough, i.e. At levels higher than the cut - off value . Menopausal status seemed to be important in terms of clinical outcome among patients bearing er+, pr+ and tff1 + tumors of histological grade ii . We found a significant difference in dfi probabilities between premenopausal and postmenopausal patients within the indicated phenotype where none of postmenopausal patients developed distant metastases during the first 3 years after the surgery (figure 4). Our result concerning postmenopausal patients suggests that the proliferation and/or migration of breast cancer cells in er+, pr+ and tff1 + tumors of grade ii are either stimulated to a low extent or not stimulated at all by higher levels of tff1 . The reason for potentially low / no stimulation of proliferation and/or migration of breast cancer cells might be due to tff1 signaling that is presumable not directly dependent on estrogen action in these tumors . It should be emphasized that patients with favorable course of the disease, as presented in figures 3 and 4, have had higher dfi probability than pn0 patients, for whom dfi probability was 0.86, while patients with unfavorable course of the disease have had lower dfi probability compared to pn+ patients, for whom dfi probability was 0.68 . Estrogen - independent tff1 signaling may be considered as responsible for a potential role of pr in suppressing tumor cell motility and/or proliferation in er - positive, histological grade ii tumors of postmenopausal patients (figure 5). Expression pathways of tff1 and pr in breast cancer may be distinct as it was observed that growth factors, such as igf - i, may increase tff1 expression while decreasing pr levels, irrespective of er levels 61 . However, the role of er (er precisely) in the regulation of tff1 expression can be overridden due to high levels of activating histone modifications in the genome . It has been reported that accumulation of these modifications at tff1 promoter may facilitate the binding of other transcription factors, rather than er, that could contribute to pathways responsible for the development of estrogen - independent breast tumors 62 . Therefore, within the group of er - positive, postmenopausal patients bearing tumors of histological grade ii, we supposed that growth factor - regulated tff1 signaling pathways (dependent or not on er) are dominant in pr - negative tumors . Considering the potential role of tff1 in promoting cancer cell migration and proliferation 19 18, the cross - talk between tff1 and pr signaling pathways may be assumed in case of tff1-positive, postmenopausal patients bearing tumors of histological grade ii . It is possible that high levels of pr suppress tumor cell motility by preventing tff1 dimerization as tff1 dimer was shown to be more potent than tff1 monomer in stimulating breast cancer cell migration 19 . High levels of pr might be also presumed to contribute somehow to a lower tumor cell proliferation . Consequently, the motility / proliferation of tumor cells is probably less suppressed in tumors of pr - negative patients . Some previous studies suggested that dual determination of tff1 and pr may be unnecessary since estrogen - dependent tumors will express both proteins . Our results are in favor of simultaneous determination of these two proteins in, at least, er - positive tumors of histological grade ii as their joint status might help to identify patients at opposite risk for development of distant metastases during the first 3 years after the surgery . In regard with above mentioned results, it should be emphasized that analysis revealed a uniform distribution of modalities of adjuvant systemic therapies among analyzed subgroups of patients (data not shown). It is noteworthy that none of 14 tff1-positive postmenopausal patients (figures 4 and 5), in addition to none of 8 tff1-negative premenopausal patients (figure 3), all bearing er - positive and pr - positive tumors of histological grade ii, developed distant metastases during the first 3 years of follow - up . This finding suggests that adjuvant systemic therapy might be omitted in case of these patients . One could argue on numbers of patients analyzed in each subgroup with regard to dfi probabilities . Although numbers are small in absolute terms, two facts should be taken into account when considering those numbers . Patients who were included in dfi analysis were supposed to meet several criteria simultaneously with respect to clinico - pathological features, i.e. Patients were stratified according to both menopausal status, histological grade, er status, pr status and tff1 status . We believe this was quite a demanding prerequisite for the analysis, leading to small numbers of patients analyzed within each subgroup . One may assume that those numbers would be greater within a much larger group of patients (a thousand or several thousands of patients). Having in mind aforementioned, our viewpoint is that numbers of patients related to each subgroup within dfi analysis are not so small in relative terms and that our results regarding dfi analysis do possess significant statistical power . Altogether, a prospective study on a much larger scale of patients is needed to validate our conclusions . In summary, our results show that tff1 expression is correlated with postmenopausal status, histological grade of tumor, er and pr status, which is in agreement with the findings of the majority of breast cancer studies on tff1 . Distribution of tff1 levels between er - positive and er - negative postmenopausal patients bearing tumors of different histological grade provided us the basis to establish the cut - off value of 14 ng / mg for distinguishing estrogen - independent from estrogen - dependent tff1 expression in breast cancer . Determination of a biologically - related cut - off value for tff1 expression represents the original contribution of this study to the field of breast cancer research . Our results regarding the clinical value of tff1 in breast cancer indicate that tff1 status might be helpful in discriminating patients at different risk for development of distant metastases within a 3-year follow - up . Within specific breast cancer subgroups, tff1 status might identify patients at high risk for relapse (tff1 + pre ii er+ pr+, tff1 + post ii er+ pr-), as well as those at low risk for relapse (tff1- pre ii er+ pr+, tff1 + post ii er+ pr+) who might not even develop distant metastases during the first 3 years of follow - up . It may be assumed that the expression and action of tff1 in breast cancer is regulated via different signaling pathways through which it stimulates motility and/or proliferation of tumor cells to a different extent . Further investigations are necessary in order to reveal the mechanism of tff1 action in breast cancer . Determination of tff1 status could assist in making decision on administering adjuvant therapy for early breast cancer patients during the first 3 years of follow - up . Tff1: trefoil factor; igf: insulin - like growth factor; egf: epidermal growth factor; tgf: transforming growth factor; ere: estrogen - response element; tre: tpa - response element; tpa: 12 - 0-tetradecanoylphorbol-13-acetate; er: estrogen receptor; ai: aromatase inhibitor; pr: progesterone receptor; dfi: disease free - interval;
In a recent issue of the new england journal of medicine, greet van den berghe and co - workers published a confirmation of the life - saving effects of tight glycaemia control by intensive insulin therapy (iit) in medical intensive care unit (icu) patients . This second study intended to answer some of the questions and criticisms raised by the landmark leuven study of 2001, which reported a 4% decrease in mortality in a surgical icu population [2 - 7]. Hopefully, these questions will be answered by the multi - centre assessments of the effects of iit, the nice - sugar and glucontrol trials, currently underway in australia and europe, respectively . Before the completion of these two indispensable studies, greet van den berghe and colleagues wanted to confirm the life - saving effects of iit in medical icu patients in order to answer specific criticisms related to the type of patients, mostly surgical with two - thirds being post - cardiac surgery patients, in the first leuven study . Indeed, patients with myocardial ischaemia could particularly benefit from a combination of a high amount of glucose and insulin, as reviewed recently . Interestingly, krinsley reported a similar reduction in mortality in a mixed population of medical and surgical icu patients . Greet van den berghe and colleagues succeeded but, as in most major contributions, their study raised more questions, which further argue for the importance of multi - centre trials ., this study cannot be considered to be positive, as the long - stayers in whom a survival benefit was found were actually not randomised . The sample of long - stayer patients (n = 767) was smaller than the calculated sample size of 1,200 patients that was required to test the working hypothesis (a 7% reduction of the absolute risk of death) with an alpha level of less than 0.05 and a beta level of 0.2 . In the entire set of patients, the intent - to - treat analysis indicated that there was actually no benefit related to iit . Moreover, mortality in the patients in whom the stay was shorter than anticipated (less than 3 days) was higher in the iit group (26.8%; 56/209) than in the conventional treatment group (18.8%; 42/224). This increase was found to be significant when analysed by the chi square test, but not by uncorrected proportional - hazards analysis, even after correcting for the difference in baseline risk factors . Compared to the first leuven study, some of the recorded secondary end points that could be considered as surrogate markers of severity (icu and 28-day mortality, requirement for dialysis, incidence of bacteraemia, requirement for prolonged antibiotic therapy, incidence of hyperbilirubinaemia and' hyper - inflammation') were not influenced as hospital mortality was . Other local factors could also have influenced the results, thereby questioning the applicability of the findings to patients managed in other icus . The mean amount of parenteral glucose infused (a mean of more than 220 g / day in long - stayers) was probably higher than in most other icus, and parenteral steroids were used in more than half of the patients . Unequivocally, these two factors reduce the risk of hypoglycaemia and the duration of episodes of hypoglycaemia, a major side effect of iit . Nonetheless, the risk of hypoglycaemia was substantial, with 25.1% of the patients in the iit group experiencing a blood glucose level below 2.2 mmol / l (40 mg / dl) at least once, compared to only 3.9% in the conventional group . The safety monitoring board of the german multi - centre study visep considered a similar increase in the incidence of hypoglycaemia important enough to stop this trial . Clearly, the safety of iit needs to be assessed in patients with significant risk of hypoglycaemia . The target ranges of glycaemia were 4.4 to 6.1 mmol / l (80 to 110 mg / dl) and 10 to 11 mmol / l (180 to 200 mg / dl), implying that there was no assessment of an intermediary blood glucose value, which is often used . Greet van den berghe and colleagues already answered this question by analysing the data of the first study and concluded that there was a dose - response effect, with the largest improvement found in patients with the lowest blood glucose level . This hypothesis was found retrospectively, however, and clearly requires confirmation from prospective trials . The use of iit in leuven is probably easier than in other institutions with a lower nurse - to - patient ratio . For an iit approach, lastly, insulin exerts many effects other than a decrease in blood glucose, which could possibly be beneficial or deleterious in different subsets of patients; these effects cannot easily be assessed and monitored in the presently available trials . In summary, questions about the efficiency and safety of iit in different icus around the world are far from answered and much work has still to be done to answer the new questions raised by the second leuven study.
To compare in - s and in - e among the three groups, the values were log - normally transformed to an approximately normal distribution before analysis, the geometric means (gm) and the geometric standard deviations (gsd) were calculated, and the steel rank sum test was applied . A single regression model and a single correlation analysis were used to evaluate the relationship between in - e and in - s . The mean age of the subjects was 38.5 years (range 2063), 87.5% were male, and 47.5% were current smokers . The mean duration of indium exposure was 8.2 years (range 0.734.7). In - e ranged from 0.004 to 24.0 g / m, and in - s ranged from 0.1 to 8.5 g / l . The gm (gsd) of in - e was 0.97 (8.68) in the smelting workers, 1.22 (2.19) in the ito workers and, 0.10 (6.49) in the other workers, respectively . The in - e values of the smelting workers and the ito workers were marginally (p=0.0709) and significantly (p=0.0069) higher than the in - e values of the other workers . The gm (gsd) of in - s was 0.93 (4.50) in the smelting workers, 0.58 (3.26) in the ito workers, and 0.12 (1.62) in the other workers, respectively . The in - s values of the smelting workers and the ito workers were significantly (p=0.011 and 0.022, respectively) higher than those of the other workers . Figure 1a shows a scattergram of log(in - e) and log(in - s). The simple regression equation was log(in - s)=0.322log(in - e)0.443, the regression coefficient was statistically significant (p=0.0002), and the simple correlation coefficient was 0.555 (95%ci 0.2900.741).fig . 1 . Scattergram of log(in - e) and log(in - s) classified by job types.the x - axis represents log(in - e) and the y - axis represents log(in - s).r: simple correlation coefficient . *: p<0.05 . Scattergram of log(in - e) and log(in - s) classified by job types . The x - axis represents log(in - e) and the y - axis represents log(in - s). Because the distribution of the in - e and in - s values was different among the three groups, scattergrams were displayed for each group (figs . The simple correlation coefficients were 0.489 (95%ci 0.4170.908) in the smelting workers (fig . Due to the small number of subjects, the distributions of the in - e and in - s values in the smelting and ito workers were not statistically significant, but they seemed to be linear (fig . As far as we know, this is the first paper concerning the relationship between in - e and in - s . Our research team has been interested in the relationship between in - e and in - s for a long time . However, we believed that it would be difficult to clarify this relationship because we believed that in - s may strongly reflect the amount of indium load in the lungs and, thus, the current in - s would not correlate to the current in - e . The reasons are supposed as follows: (1) due to the hardly soluble characteristics of indium compounds, the clearance rate of accumulated indium compounds in the lungs is very small7; (2) the in - s levels of formerly indium - exposed workers do not readily decrease4 and the biological half - life of in - s in formerly exposed workers who perfectly removed from indium exposure is around 8 years8; and (3) many indium processing plants have instituted labor hygiene management systems, and in - e and indium inhalation concentration may not be parallel due to the wearing of effective protective devices6 . In this study, we had a chance to measure in - s and in - e in 11 plants that had not yet taken action to improve the work environment . Workers in these plants wore no or ineffective respiratory protective devices, and the in - e levels were expected to be low, so the indium load in the lungs of the workers could be expected to be small . However, it should be noted that extrapolation of this study s results to other indium - exposed populations requires careful consideration of whether the indium lung load in the population is actually negligible and whether the population wears appropriate respiratory protective devices . After the log transformation of in - s and in - e, this suggests that in - s may increase in - e - concentration - dependently in the workers in this study . Figures 1b, 1c and 1d seem to suggest that the relationship between in - e and in - s may vary due to the chemical form of the indium compounds . Ito, indium trioxide, indium metal, or indium alloy was the major chemical form in the ito workers, the smelting workers9, and the other workers . Though the number of workers was very small, in - e was correlated to in - s in the ito workers and in the smelting workers, but not in the other workers . We have no information about the difference in the kinetics of each chemical form in the lung, but indium metal or indium alloy may show different kinetics unlike ito or indium trioxide . First, the number of study subjects was not sufficiently large, and information of relatively high in - e levels was lacking . This may make it difficult to smoothly interpret the results, and indicates a need to expand the study . Secondary, day - to - day variations of in - s are considered to be negligible, but day - to - day variations of in - e cannot be ignored . We may need to measure in - e on multiple days . In conclusion, in - e and in - s seem to be positively correlated, but more data are needed to draw final conclusions . Studies of the kinetics in the lungs of each indium chemical form are also necessary to elucidate the relationship between in - e and in - s . This study was supported by grants - in - aid for scientific research (project no . 24590758 and 26860443) from the ministry of education, culture, sports, science and technology of japan (2012 - 13 and 2014 - 2015).
Distal gastrectomy is a well - known risk factor for developing gastric stump cancer later . In particular, cancer frequently occurs near the gastrojejunostomy site in the remnant stomach following a billroth ii reconstruction . One proposed theory is that the enterogastric reflux of bile and pancreatic secretions chronically irritate the gastric mucosa promoting neoplasia . Gastric neuroendocrine carcinoma (nec) is an uncommon tumor with a strong malignant potential and an extremely poor prognosis . In the present case report, we describe a patient who had undergone ge with braun enteroenterostomy (bee) 50 years prior to presentation and had developed a mixed adenoneuroendocrine carcinoma (manec) at the anastomotic site . A 75-year - old man was diagnosed with gastric cancer in june 2012 at showa university fujigaoka hospital . Fifty years previously, he had undergone a gastric bypass operation with a loop gastrojejunostomy to treat stenosis caused by a duodenal ulcer . His medical history included chronic renal failure (managed with hemodialysis), right idiopathic pleural effusion, third degree av block (managed by a pacemaker), and myelodysplastic syndrome . As of 2006, chronic gastric hemorrhage had been observed at the ge anastomotic site, and endoscopy was performed . In june 2012, an upper gastrointestinal endoscopy revealed a slightly irregular mucosa surrounded by a smooth elevated area at the stomal site . Ct showed an enlarged lymph node on the lesser curve of the stomach, which is suggestive of metastasis . However, the gastric wall did not appear thickened, which can occur secondary to neoplasia, and there was no indication of distant metastasis . The cancer was diagnosed as t2 n1 m0 stage iib according to the tnm classification . Since the patient already had poor systemic function, surgery carried a very high risk of morbidity and mortality . The surgeon repeatedly discussed the treatment options with the patient who ultimately elected surgical excision to avoid potential obstruction and hemorrhage at the stomal site . In october 2012, a distal gastrectomy, a small intestinal partial resection (afferent and efferent loops) and a roux - y reconstruction were performed . During the laparotomy, hepatic sclerosis and ascites the significant collateral vasculature and the strong adhesion between the stomach and the liver and between the jejunum and the transverse colon from the previous ge made surgery difficult . Numerous firm and small lymph nodes were identified in the lesser omentum and suspected to be metastatic . A small intestinal partial resection, including the bee site, was performed followed by a distal gastrectomy . Metastasis was observed in the common hepatic arterial lymph nodes similar to the metastasis observed in the para - aortic nodes, and a curative resection was deemed impossible . The surgery lasted 6 h, and there was a 1,492-ml intraoperative hemorrhage . Immediately after transferring the patient to the intensive care unit, he suddenly stopped breathing, and resuscitation was initiated . A 5 4 cm type-5 lesion was identified in the posterior wall at the stomal site on gross pathologic examination . 2) showed that the tumor histologically comprised two elements: a tubular adenocarcinoma on the gastric side and nec - like cells in a typical mass lesion comprising infiltrating nests of small uniform tumor cells on the jejunal side, which made up 40% of the tumor . 3) showed that the nec - like cells were positive for neural cell adhesion molecules and partially positive for synaptophysin and somatostain receptor type 2, but negative for chromogranin a. the ki-67 labeling index was> 20% . The final pathological diagnosis was manec, according to the 2010 who classification system for digestive neuroendocrine tumors . Since the first reports of carcinoma developing at the gastric remnant after gastrectomy appeared in the 1950s, numerous cases have been reported . Notably, patients who underwent a billroth ii gastrectomy developed stump carcinomas most frequently in the anastomotic area, whereas in patients who underwent a billroth i gastrectomy, neoplasia was more frequently located outside the surgical stump . A late development of stomal gastric cancer has also been reported following ge without concurrent gastric resection . A prior study of anastomotic carcinoma revealed that the tumor rarely invades the jejunum at the stoma . Persistent secondary bile reflux promotes chronic gastritis and eventual metaplasia, which is a likely precursor of neoplasia . Nishidoi et al . Hypothesized that duodenogastric reflux causes the development of remnant gastric carcinoma in mnng - treated rats . In a clinical study by tanigawa et al ., p53-positive cells increased at the anastomosis site of billroth ii procedures, which may reflect increased dna damage secondary to active gastritis . In 1893, braun introduced the enteroenterostomy anastomosis between the afferent and efferent limbs immediately distal to a ge . Using radionuclide biliary scanning, vogel et al . Found that bee adequately diverts a substantial amount of bile from the stomach in patients undergoing ge or billroth ii resection . This would presumably prevent stomal cancer development secondary to bile reflux . However, using a rat model, wieman et al . Found that the diversion of duodenal secretions away from the ge did not alter the carcinoma incidence . Similarly, in a clinical study of 22 japanese patients who developed stomal cancer after ge, ichikawa et al . Reported that bee did not prevent cancer development in the 10 patients who underwent the procedure . In our case, stomal cancer could also not be prevented by bee . Gastric nec is an uncommon tumor, which reportedly comprises 0.10.6% of gastric cancers and typically possesses adenocarcinoma components . Definitive diagnosis of mixed nec by endoscopic biopsy is thought to be difficult; in previous reports, the adenocarcinoma portion covered the surface mucosa, while the nec developed in the submucosa and deeper layers . Indeed, although our patient underwent annual endoscopies yearly, his anastomotic nec was quite advanced at the time of diagnosis . According to the 2010 who classification system, neuroendocrine neoplasms of the digestive system are a diverse group that includes neuroendocrine tumors, which are well differentiated and graded according to their proliferative activity as g1 or g2 as well as necs, which are poorly differentiated and graded as g3 . Adenocarcinoma components constituted> 30% of the tumor in our case, fulfilling the criteria for a manec as defined by the who classification . The prognosis of patients with gastric nec is extremely poor because the tumor behaves aggressively and frequently metastasizes to the liver and lymph nodes, even during the early stages . This particular case of nec at a ge anastomotic site is the first known report in the english literature . In japan, 6 cases with various diagnoses of stomal necs, including neuroendocrine cell carcinoma, endocrine cell carcinoma, and small cell carcinoma, have been reported so far (table 1) [14, 15, 16, 17, 18, 19]. These cases were diagnosed by histochemical neuroendocrine markers but were not evaluated according to their proliferative activity . The ki-67 labeling index is the most characterized proliferation - associated marker and has been proposed as an important parameter in defining necs . In 5 of the aforementioned japanese patients who underwent ge without bee, the duration between initial surgery and stump carcinoma diagnosis ranged from 20 to 47 years . Bee was performed only in the present case, which had the longest duration between initial surgery and cancer development . Except for case 6, the patients diagnosed with stomal nec had a short survival time lasting less than 1 year . Histopathological examination revealed that the gastric portion was an adenocarcinoma, while the duodenal portion was a nec . The nec component was clearly distinguishable from the pyloric ring adenocarcinoma, not only at the anatomic junction, but also at the environmental border between these two organs . Similarly, in our case we observed an nec on the jejunal side of the tumor and an adenocarcinoma inside the tumor . Previous studies have reported on neuroendocrine differentiation in the gastric adenocarcinoma . In a genome - wide loh study, kim et al . Concluded that most mixed glandular gastric necs evolved from a glandular precursor prior to neuroendocrine differentiation, and occasionally, dual differentiation concurrently arose from a single precursor . We surmise that in our case, the constant exposure to secondary bile may have induced a gastric mucosal adenocarcinoma, which finally differentiated into a nec . The aforementioned japanese cases were mostly pure nec, and the mixed type was only observed in one case . Eventually, the remaining adenocarcinoma portion in our case might have been replaced by the nec.
For decades, urinary sodium (nau) was used to define the presence of structural damage to the kidneys in the setting of oliguria or azotemia . The preserved capacity of the tubules to retain sodium was the physiological basis to interpret low levels of nau as a functional response to a low renal perfusion state: socalled' pre - renal' azotemia . The loss of this capacity by the kidneys was considered a marker of' acute tubular necrosis' (atn). In the past, levels of nau below 20 meq / l were considered markers of pre - renal impairment and above 40 meq / l as markers of intrinsic renal disease . Recently,' pre - renal' and' atn' paradigms have been frequently criticized: first, because many cases classified as atn lack this finding in histopathological studies; and, second, because increasing knowledge of acute kidney injury (aki) revealed a dissociation between renal hemodynamics and nau, especially in sepsis . Therefore, these old paradigms gave place to a new paradigm: that nau is useless as a tool in aki management . The aim of this commentary is to question if this new paradigm should be sustained . In 2006, a systematic review revealed that nau values were widely variable within and between studies with no consistent values to distinguish normal kidney function, pre - renal azotemia and atn . A contemporaneous experimental study inducing hyperdynamic sepsis revealed progressively lower levels of nau . It was hypothesized that the sodium retentive state was due to loss of glomerular filtration pressure . The authors concluded that nau was not a reliable marker of renal perfusion (breaking the old' pre - renal' paradigm). Since then, a new paradigm has emerged: nau must not be used as a diagnostic tool in aki . This phenomenon is the possible explanation for apparently paradoxical increases in the sublingual tissue partial pressure of carbon dioxide (pslco2; a marker of microcirculatory stagnation) in parallel with increasing cardiac output during sepsis . Hence, a similar phenomenon could explain the paradox between an increased renal blood flow and low nau levels . Glomerular perfusion pressure, not total renal blood flow, is the main determinant for nau levels . In inflammatory states, low glomerular perfusion pressure may occur in the presence of increased renal blood flow, with activation of sodium - retaining mechanisms . Although tubular injury is an early event in aki, most studies still found low fractional excretion of sodium levels in this context . We may conclude that too much injury is needed to impair the global tubular capacity to retain sodium . Recent unpublished results by our group also suggest that sodium retention is progressively more intense with increases in aki severity except in very advanced stages (aki network stage 3); we hypothesize that extensive tubular injury jeopardized sodium reabsorption . In our findings, such progressive decreases in nau began earlier than increases in creatinine, as described in a case report . First, nau is measured only once instead of sequentially; as previously demonstrated, nau responds fast to acute hemodynamic alterations so that relative alterations in it may be more relevant than an isolated nau value . It is important to remember that nau has a very large physiological range that depends on numerous variables . Of these, the most likely responsible for an abrupt decrease in nau value is a decrease in glomerular filtration rate . Second, nau is still treated as a categorical variable; the dynamism of nau is lost if nau is viewed as' <20 meq / l' or'> 40 meq / l' . Third, nau is usually assessed only in the presence of oliguria or azotemia . In a recent article, we suggested that urinary electrolyte measurement may alert for the presence of aki development before increases in creatinine or oliguria . In that study, patients who developed aki in the first 4 days after admittance to the icu had significantly lower nau values at admission . Low nau values in aki can be a sign of microcirculatory impairment in the kidneys . We have observed many critically ill patients with very low nau levels on the day that renal replacement therapy was initiated . This is not surprising in the context of multiple organ failure, which may be caused by systemic microcirculatory failure . From this perspective, the lower the nau, the greater the microcirculatory stress . On the other hand, including 10 healthy volunteers, the mean nau was 104 48 meq / l . Meq / l at icu admission in patients who did not develop aki during the study period . However, high nau values can be found in patients with aki receiving diuretics or in advanced aki stages . As for many other monitoring parameters in critical care medicine, the first step in defining nau utility in daily practice is to understand properly what it is saying to us and in which contexts . Aki: acute kidney injury; atn: acute tubular necrosis; nau: urinary sodium.
In patients receiving radiotherapy and chemotherapy for esophageal and lung cancers, it is not uncommon to witness development of life - threatening tracheo - esophageal fistula (tof). Various procedures have been tried including surgical repair, endoprostheses, endo - bronchial gluing and tracheal stents . Other workers have shown good long - term results of palliative treatment with covered expandable metallic stents . We report a case of ca esophagus who after chemotherapy and radiotherapy developed to fistula; esophageal stenting was done but patient was still having cough and dyspnoea . Fibreoptic bronchoscopy (fob) showed a large rent in the posterior wall of the trachea with marked narrowing of the tracheal lumen because of protrusion of the esophageal stent . He was successfully managed with the insertion of an ultraflex - covered tracheal stent and achieved relief from cough and dyspnoea . A male patient, ks, aged 60 years, suffering from esophageal cancer was electively operated upon in october 2007 after discussion in the multispecialty tumor board . The gross specimen was an ulcero - proliferative growth measuring 4.5 1.7 cm in size located 2.3 cm from the gastro - esophageal (ge) junction and was reported to be moderately differentiated squamous - cell carcinoma . Computed tomography (ct) chest and abdomen showed esophageal gastric anastomosis in the lower neck with irregular mass abutting the posterior and left wall of the trachea and encasing the left carotid artery . Ultra sound (usg) and ct - guided fine needle aspiration cytology (fnac) from liver nodules showed metastatic deposits of poorly differentiated squamous - cell carcinoma . He received three cycles of tip- (paclitaxel / ifosfamide / cisplatin) based chemotherapy and palliative external beam radiotherapy to the l2s1 spine and pelvis with a dose of 30 gy/10 #during june 2010 . Bone scan in july 2010 showed increased uptake in the sternum, l2 vertebra, bilateral acetabulam and bilateral iliac bones . In the month of august 2010 ugi endoscopy on 19 august 2010 showed an esophageal growth starting at 16 cm from upper esophageal sphincter (ues) and extending up to 23 cm with tof . His x ray chest was clear and the esophageal stent was seen in place . Bronchoscopy was advised and done as per american association for respiratory care (aarc) criteria . There was a huge gap in the posterior wall of the trachea with esophageal stent visible and protruding into the trachea and narrowing the lumen [figure 1]. As the patient was severely symptomatic and the situation could have progressed to complete obstruction of the trachea, tracheal stenting was done on 31 august 2010 . Guide wire (zebra) was passed through the working channel of the bronchoscope and bronchoscope was withdrawn keeping the guide wire in trachea . A 10-cm covered ultraflex tracheal stent [figure 2] was passed over the guide wire and was deployed in proper position [figure 3] covering the tof . The whole procedure was done under vision by keeping the bronchoscope inserted through the left nares . He was discharged after two days on 2 september 2010 and is doing well four months after the procedure . Check bronchoscopy was performed four months after the tracheal stent placement and there was no migration or any other complication of the stent, except that the tracheal lumen had slightly narrowed down from the posterior side . Today they are indicated for re - establishing patency of compressed or strictured central airways, supporting weakened cartilages or sealing of fistulas . Stents are divided into four groups: polymer stents such as dumon stents, metallic stents such as palmz stents or uncovered wall stents, covered metallic stents such as the covered ultraflex stent, hybrid stents such as dynamic stents . Polymer stents such as dumon stents, metallic stents such as palmz stents or uncovered wall stents, covered metallic stents such as the covered ultraflex stent, hybrid stents such as dynamic stents . Airway and/or esophageal stent insertion provides an effective approach to improve the quality of life (qol) in patients with malignant tof . Stents can be placed bronchoscopically in the airways and with endoscope in the esophagus to seal the defect and restore the patency of passages with resumption of oral feeds . Although single tracheal or esophageal stents were successful in 91% of cases, 9% needed double stenting of both trachea as well as esophagus . Our patient was in this category who did not remain symptom - free with esophageal stent alone and needed both esophageal and tracheal stents . Nowadays, in cases of severe esophageal and tracheal obstructions, the pulmonologists are involved early and recent reports suggest double stenting to be a promising option . There is no doubt that double stenting is the gold standard and should be practiced but the present case was under care of the oncologist and as the predominant symptom was dysphagia he called upon the gastroenterologist who performed endoscopy and on finding tof informed the oncologist . The oncologist explained the huge financial burden of double stenting (50,000 to 60,000 inr for each stent) and deferred the tracheal procedure . Anyhow, the tracheal stenting was done not too late (done within 10 days and during the same admission). The paper highlights and infers about the importance of this controversy . This leads to frequent aspiration, the most frequent sign of tof being coughing after swallowing; without prompt palliation, death occurs rapidly, with a mean survival time of between one and six weeks in patients who are treated with supportive care alone . Herth et al ., have reported a mean survival of 252.9 days with combined airway esophageal stent . Our patient was successfully taking food and was not dyspnoeic four months after the stent placement . However, up to 20% of silicone stents have been reported to migrate, with resultant reocclusion . Other problems of silicone stents include clinically significant stent obstruction with inspissated secretions due to impairment of mucociliary clearance, impedance of the cough reflex, lack of flexibility in the airway, high mucosal pressure and higher chance of bacterial colonization . Initially, to deploy the silicon stent, patients were administered general anesthesia and the procedure was done with rigid bronchoscope under fluoroscopic guidance . Nowadays, these stringent requirements have been relegated to the past and large tofs are successfully sealed by deployment of self - expandable stents through flexible bronchoscope without the need of fluoroscopy . The primary advantage of the expandable metallic stents is their easy delivery by means of flexible bronchoscope under topical anesthesia and their conformability to the airway anatomy due to their self - expanding characteristics . With the use of self - expandable metallic stents (sems), there is an increase in the qol in patients with inoperable esophageal and lung tumors . We have used a covered sems which has an advantage over plastic or silicon stent not only in covering large defects, but also in having lower rates of migration along with adequate maintenance of patency of the passages . Our patient had an increase in dyspnoea after placing the esophageal stent; a similar phenomenon has been reported by nomori et al ., their two patients suffered from respiratory distress after placement of the esophageal stent because of compression of the trachea from the posterior wall . They have further observed that for patients with both esophageal and tracheobronchial stenoses, a stent should be introduced into the tracheobronchus first . The success rate for closure of tof using endoscopic methods varies from 87 100% . On repeat bronchoscopy, our patient was comfortable, his stent was placed at the right position and there were no complications . In the large series by herth et al ., none of their patients developed stent migration or required repositioning of the stent and there were no complications of perforation or mediastinitis . The american college of chest physicians guidelines for palliative care in patients with a malignant tof recommend stent insertion in both the tracheobronchial tree and the esophagus for symptomatic relief . To conclude, we report a case of ca esophagus post surgery who underwent chemotherapy and radiotherapy and later developed to fistula . Initial placement of an esophageal stent did not give him much of a reprieve and later, tracheal stenting helped him to get complete relief from his disabling symptoms.
Castleman's disease is a lymphoproliferative disorder with benign hyperplastic lymph nodes characterized histologically by (a) follicular hyperplasia, and (b) capillary proliferation with endothelial hyperplasia (1). This disease has been classified histopathologically as hyalinevascular, plasma cell, or a mixed type variant of the two (2, 3). The plasma cell and mixed types are often associated with' multicentric castleman's disease' (mcd), which shows various systemic manifestations, such as fever, anemia, hypergammaglobulinemia, hypoalbuminemia, and an increase in acute phase proteins (2). Mcd is often refractory to conventional therapeutic strategies, such as corticosteroid, cytotoxic agents, and/or radiation (4, 5). The frequency of mcd associated with a lung lesion among the japanese population is high (~70%) (6) and such progressive lung lesions often lead to death (2, 7). Interleukin-6 (il-6) is a pleiotropic cytokine with a wide range of biologic activities, such as hematopoiesis, regulation of immune responses, and inflammatory responses (4). Patients with the plasma cell type castleman's disease often generate large quantities of il-6 in the germinal centers of hyperplastic lymph nodes (8).' Tocilizumab' is a humanized anti - interleukin-6 receptor antibody that neutralizes the pleiotropic actions of il-6 . It was approved for use in japan for the treatment of castleman's disease in 2005 . This report describes a case of mcd with an associated lung lesion, which responded dramatically to tocilizumab in combination with corticosteroid and tacrolimus . A 43-yr - old female visited a nearby hospital because of abnormal shadows including multiple nodules and reticular shadows on chest radiography found at an annual medical checkup in 2005 . They were unable to obtain a biopsy specimen from one of the nodules in s10 of the left lung because the patient had a strong bleeding tendency . She was referred to this hospital for further examination on june 30, 2005 . On admission, the laboratory data were: erythrocyte sedimentation rate, 119 mm/1 hr; white cell count, 8,900/l; hemoglobin, 8.4 g / dl; platelet count, 39.410/l; serum aspartate aminotransferase, 22 iu / l; alanine aminotransferase, 22; total protein, 9.7 g / dl; albumin 2.6; creatinine, 0.48; pt, 13.1 sec (inr 1.45); aptt, 39.6 (control, 10.4); fibrinogen, 750 mg / dl; kl-6, 277 u / ml (reference range <500); c - reactive protein, 11.7 mg / dl (<0.3); serum immunoglobulin (ig)g, 4,570 (870 - 1740); iga, 491(110 - 400), igm, 706 (35 - 220), ch50, 52.3 u / ml (30 - 50), soluble il-2 receptor, 1,400; serum il-6, 6.8 pg / ml (<4); rheumatoid factor (rf), 1,330 iu / ml (<20). Autoantibodies, including antinuclear antibody, anti - ds - dna, anti - sm, anti - rnp, cytoplasmic antineutrophil cytoplasmic antibody (anca), and myeloperoxidase - anca were all negative . Chest ct scan disclosed a slight enlargement of the mediastinal lymph nodes, centrilobular nodules, thin - walled cysts, the thickening of the bronchovascular bundles, and ground - grass opacities, all of these findings were compatible with those of lymphocytic interstitial pneumonia (lip; fig . Gallium citrate scintigraphy did not reveal any evident accumulation . A lung surgeon and a thoracic physician declined to perform a lung biopsy because of her bleeding tendency (bleeding time: 6 min 30 sec) and poor general condition . A biopsy of an inguinal lymph node was obtained for making a definite diagnosis (fig . She was diagnosed with mcd and undifferentiated arthritis based on the characteristic pathology of the specimen of the inguinal lymph node, ct findings of the bilateral lung lesions and laboratory data . She was initially administered intravenous corticosteroid (methylprednisolone, 500 mg / day, 3 consecutive days) followed by oral corticosteroid (methylprednisolone, 16 mg / day), and, 375 mg / m rituximab every week for 4 weeks . The polyarthralgia instantly disappeared; however, none of the other clinical and laboratory parameters were fully resolved . Therefore the therapeutic regimen was changed to tocilizumab (8 mg / kg, every 2 weeks), oral corticosteroid (methylprednisolone, 16 mg / day) on october 25, 2005 . The patient requested that the interval between tocilizumab to be increased to more than every two weeks . The dose of corticosteroid in combination with tocilizumab should be kept as low as possible to avoid various side effects, such as osteoporosis, hyperglycemia, and hypertension . Both the possible merits and demerits of the drug were explained to the patient and her oral consent was obtained . Laboratory findings, including anemia, hypergammaglobulinemia, and an increase in acute phase proteins responded to this regimen (fig . The enlargement of mediastinal lymph nodes and abnormal shadows were also partially alleviated (fig . The intervals of the tocilizumab - administration sessions were extended from every 2 weeks to every 3 weeks from january 2006 . Oral corticosteroid was gradually tapered from 16 mg / day finally to 4 mg / day . The treatment of mcd includes various therapeutic strategies such as corticosteroid, chemotherapy, rituximab, or tocilizumab . Rituximab, an immunoglobulin g1 (igg1) monoclonal antibody to cd20, has the potential for b - lymphocyte depletion via antibody - dependent cell mediated - cytotoxicity (adcc), complement dependent cytotoxicity (cdc), and apoptosis . Kaposi's sarcoma - associated herpes virus (kshv / hhv8) was thought to be associated with the development of castleman's disease, especially in patients infected with human immunodeficiency virus (hiv) and rituximab was reported to be effective in hiv - related castleman's disease (11). The effectiveness of rituximab was limited in the present case because the patient was not infected with hiv . A humanized anti - interleukin-6 receptor antibody, tocilizumab, was shown to be effective for the treatment of mcd in an open label trial, and has been widely used in japan (5). The frequency of mcd associated with a lung lesion is relatively low (10 - 20%) in the united states . In contrast, nishimoto et al . Reported that 18 of 28 japanese cases (= 64.3%) had a lung lesion (6). The ct findings of intrathoracic involvement in mcd include bilateral hilar and mediastinal lymphadenopathy, centrilobular nodular opacities, thin - walled cysts, interlobular septal thickening, and ground - glass attenuation (9). Some reports have noted that the lung lesion in mcd is compatible with lymphocytic interstitial pneumonia (lip) (5, 9). (12) reported a case of mcd with a lung lesion that responded to tocilizumab . (7) described that the administration of tocilizumab for 2 yr had no effect on the lung lesion in an mcd patient . Il-6 production by the germinal center b cells in the swollen lymph nodes of patients with mcd is remarkable (8), but the roles of t cells are unknown . If t cells do participate in the pathogenesis, immunosuppressive drugs for t cells should be useful . In fact, mcd, which is classified as a lymphoproliferative disorder, has characteristics of an autoimmune disease, demonstrated by the production of autoantibodies . Several studies have shown that these drugs are active not only against t cells but also against other cells . They suppress the promotion of il-6 in the rheumatoid synovium where t cells are relatively scarce (13). They also suppress il-6 production in monocytes, and tnf- production in b cells (14, 15). Pham et al . (16) reported that inhibition of nf-b and nfat activation in aggressive b - cell lymphomas by calcineurin inhibitors suppressed the cd40 ligand expression in b cells and lymphoma cell survival in an in vitro experiment . Some of these actions of tacrolimus, if not all, may be associated with the efficacy of the drug for the treatment of mcd . (17) reported that non - cytostatic immunomodulatory therapy including corticosteroid, cyclosporine a and thalidomide treatment was effective for castleman's disease . The long - time use of tocilizumab and corticosteroid is necessary for the treatment of mcd . Corticosteroid has various dose - related side effects, and acquisition of drug resistance possible . P - glycoprotein (p - gp) plays a pivotal role in the latter . Peripheral lymphocytes in patients with autoimmune diseases could express p - gp on their cell surface . Calcineurin inhibitors have an antagonistic activity to p - gp: they may inhibit corticosteroid - resistance in peripheral lymphocytes in vivo (18, 19). The dose of corticosteroid could be tapered in the current case, at least in part, due to the antagonistic activity to p - gp of tacrolimus . In conclusion, this is the first report to describe that tocilizumab, in combination with corticosteroid and tacrolimus, shows a dramatic effectiveness in the treatment of a lung lesion in an mcd patient.
Bladder pain syndrome / interstitial cystitis (bps / ic) is a clinical diagnosis primarily based on symptoms of urinary urgency, frequency, and suprapubic or pelvic pain . The prevalence of bps / ic ranges from 10 to up to 510 cases per 100,000 population . The etiology of bps / ic is so far unknown, with several theories under discussion, including an autoimmune response, mast cell activation, neuropathic changes, occult infection, toxic substances in the urine, and a primary defect in the glycosaminoglycan (gag) layer of the bladder mucosa . Prominent among the theories is that bps / ic may be related to a primary defect of the gag layer of the bladder urothelium . The major classes of gags include hyaluronic acid, heparin sulfate, heparin, chondroitin 4-sulfate and chondroitin 6-sulfate, dermatan sulfate, and keratan sulfate . Hyaluronic acid contributes an important proportion of the gags of the bladder surface and is considered a good candidate for gag substitution in patients with bps / ic . Gag substitution therapy with hyaluronic acid has been shown to benefit patients by relieving the distressing symptoms of pain, urinary frequency, urgency, nocturia, and hematuria . For intravesical hyaluronic acid therapy, several uncontrolled studies using 40 mg dissolved in 40 ml of normal saline solution weekly for 4 to 6 weeks and then monthly thereafter have reported response rates varying from 71% to 30% in bps / ic as well as in other chronic inflammatory bladder diseases such as radiation and recurrent bacterial cystitis . However, like the other treatment options for patients with bps / ic, intravesical hyaluronic acid therapy has no long - term efficacy and its benefits on bps / ic symptoms decrease in 24 weeks . On the other hand, up to 70% of patients treated with intravesical hyaluronic acid several studies have evaluated the bladder distension with electromotive drug administration (emda) in patients with bps / ic, and the findings of these studies suggest that office distention with emda is a viable alternative for select ic patients ., results in patients with t1 bladder cancer improved in patients administered bacille calmette - gurin (bcg) and mitomycin via emda compared with the group administered bcg alone . Electromotive mitomycin increases tissue uptake compared with passive diffusion . Given that instillation of hyaluronic acid via emda may increase tissue uptake and improve efficacy, we designed the present randomized prospective study to evaluate whether intravesical hyaluronic acid installation with emda would improve the response rate and long - term efficacy of the therapy . Within the time period of 2004 - 2005, 31 patients who had been diagnosed with bps / ic were examined . Inclusion criteria were as follows: all patients fulfilled the national institute of diabetes and digestive and kidney diseases criteria for bps / ic, no other treatments were allowed during the study, and patients had been evaluated for urinary tract infection and it had been ruled out . Exclusion criteria were as follows: any other medication for ic during the study period, neurogenic bladder, history of pelvic surgery or trauma to the pelvic region, presence of active urinary tract infection, frequency of urination less than 8 times / d, presence of bladder or lower ureteral calculi, presence of benign or malignant bladder tumors, presence of active genital herpes infection, or presence of chemical or radiation cystitis . A, patients were treated with 40-mg intravesical hyaluronic acid (cythyal, bioscience gmbh, pontiac, mi, usa) administered via a hydrophilic 12-fr foley catheter and were instructed to retain the installation volume for at least 60 minutes . In group b, patients were catheterized with a 16-fr (ce - das orugenics / ag 9701, mirandola, mo, italy) catheter (special catheter for emda with a spiral silver electrode in the first part), and 40-mg hyaluronic acid in 40-ml saline was instilled into the bladder by gravity . Two patch electrodes were placed in the suprapubic region by using an ample amount of contact gel to avoid burns . The electrodes were taped tightly in place to achieve good contact and to prevent dispersion of current over the abdomen . Emda was then performed by using the current generator with the following parameters: polarity positive, rise rate 30 - 60 ma / s, peak current 60 ma, pulsed output, and treatment time 25 minutes . In both groups, instillations were performed weekly in the first month and then monthly after 2 months . Urine analysis was performed for all patients and active urinary tract infection was ruled out . All patients followed the same diet controls (no caffeine, alcohol, or beverages that might acidify the urine, such as cranberry juice or orange juice) during their treatment and follow - up . Potassium sensitivity test performed in all patients before treatment . Cystoscopy with hydrodistention was performed before treatment in all patients and the presence of hunner ulcers was noted . Baseline voiding symptoms were recorded in 2-day voiding diaries, a visual analogue scale (vas) was used to assess worst pain, and ic symptom and problem indexes were used to assess ic . At visits at 1, 6, 12, and 24 months, the patients returned 2-day voiding diaries, marked the vas score, filled out the symptom and problem questionnaires, and gave the global response assessment (gra). The gra was defined as follows: 1, worse; 2, no change; 3, slightly better; 4, moderately better; 5, much better; and 6, completely cured . The parameters used for assessment of treatment response were based on the study by propert et al . . The physicians who assessed the treatment response were blinded to the patients' means of application of hyaluronic acid . The primary end points of the study were vas score, gra, and micturition frequency in 24 hours . Secondary end points were mean voided volume, number of nocturia episodes, and ic symptom and problem scores . Randomization was performed by the block randomization method and ncss software (ncss, kaysville, ut, usa) was used . Sample size was estimated as 25 with a power of 0.80 and an effect size of 50% (3 points of decrease in vas score). Baseline factors were compared with the t - test and differences between treatment outcomes were calculated with the student t - test . The paired - samples t - test was used to compare treatment results of a single group in certain time intervals . Multivariable cox proportional hazards regression was performed to determine any factor predictive of treatment response . Of the 31 patients enrolled, 25 patients were female and 6 patients were male . There were 15 (3 males and 12 females) patients in group a, and 16 (3 males and 13 females) patients in group b. there were no significant differences in baseline characteristics between the groups (table 1). During cystoscopy, hunner ulcers were observed in 3 and 4 patients in groups a and b, respectively . Oral pentosan polysulfate had been prescribed to 6 patients in group a and 9 patients in group b previously and those patients did not respond to pentosan polysulfate . Also, 8 patients in group a and 8 patients in group b had a history of intravesical heparin therapy . None of the patients experienced any serious adverse events and no patients refused treatment related to side effects . Treatment responses in both groups were compared at certain time intervals and are summarized in table 2 . Both treatment arms were successful at months 6 and 12 compared with baseline in all parameters, but group b was found to have statistically better results in those given time intervals . However, both groups were found to have no statistically significant improvement at month 1 compared with baseline . At month 24, voiding frequency and vas were the only parameters that were significantly improved compared with baseline in both groups, and there were no statistically significant differences between the groups . The results of the multivariable cox proportional hazards regression showed that treatment with emda, positive kcl test result, and voiding frequency> 17 were associated with higher response rates . The other parameters involved in the multivariate analysis were not found to be associated with higher response rates . Because of its uncertain cause and pathogenesis, there is currently no cure for bps / ic and the goal of therapy is symptomatic relief . Improvement can be attained with several treatment options, including oral therapy (e.g., pentosan polysulfate sodium), intravesical therapy (e.g., gag substitution therapy with specific agents), and even surgical interventions, although relapses are common . Gag substitution therapy with hyaluronic acid in patients with bps / ic has been evaluated in previous studies . Explaining the mechanism of hyaluronic acid treatment in patients with bps / ic as just the reconstitution of a morphologically defective gag layer may not be exactly right . Hyaluronic acid has various biological activities including inhibiting the adherence of immune complexes to polymorphonuclear cells, marked inhibition of leukocyte migration and aggregation depending on viscosity, regulation of fibroblast and endothelial cell proliferation, enhancement of connective tissue healing, and creation of a barrier over membranes, thus regulating movement of solutes . All of the biological activities listed above may contribute to the action of hyaluronic acid in interstitial cystitis . Reports in the literature of treatment results with long - term follow - up differ . In a study by morales et al ., the complete or partial response rate of intravesical hyaluronic acid treatment for up to 1 year was 71%; however, beyond week 24 there was a moderate decrease in the response rate of the medication . In a danish open and uncontrolled pilot study, 20 patients with bps / ic received weekly bladder instillations of hyaluronic acid for 1 month and monthly instillations for an additional 2 months . The patients were then offered further monthly instillations, and all patients were evaluated after 3 years . In contrast with the morales et al . 's study, the danish study showed long - term efficacy of intravesical hyaluronic acid therapy . Three years after the initiation of treatment, 65% of patients (13 of the 20 patients) still responded to treatment in terms of symptom remission and pain score reduction . The patients in the danish study received a longer duration of intravesical hyaluronic acid therapy (monthly for 3 years vs. 1 year). We think that the difference in these long - term efficacy results may be due to the longer duration of intravesical hyaluronic acid in the danish study . Treatment results with long - term follow - up were also recently reported by engelhardt et al . . In that study, 24 of the 48 patients were evaluated after 5 years, and 20 of these patients reported decreased pain . Several studies have reported that installation of the agent into the bladder with emda improves the treatment efficacy in patients with bladder cancer and bps / ic . Compared mitomycin delivery between passive diffusion and electromotive administration in high - risk patients with superficial bladder cancer . Peak plasma concentrations of mitomycin were 5.5 times higher in patients who underwent electromotive delivery than in those who underwent passive diffusion, which suggests that the cells of the bladder wall were exposed to incremental concentrations of this magnitude . The median time to first recurrence of a tumor was nearly doubled with electromotive delivery compared with passive diffusion and was similar to bcg alone - a third comparator group . In another study by di stasi et al ., the investigators randomly assigned 212 patients with pathological t1 bladder cancer to bcg alone or bcg and electromotive mitomycin . They concluded that the bcg and electromotive mitomycin group had significantly fewer progressions and deaths from bladder cancer . Performing emda in patients with lidocaine and dexamethasone installation with emda followed by cystodistention in 21 women showed a 25% success rate up to 6 months after instillation . Using a similar technique, riedl et al . Noted complete resolution of bladder symptoms in 61% of patients (8 of 13). In a study by rose et al ., bladder distention with two different anesthetic strategies was evaluated: simple instillation of alkalized lidocaine and emda of lidocaine . Those authors concluded that lidocaine emda is superior to alkalized lidocaine in that it allows for a greater distention of the bladder for a longer period of time . In another study by the same group of authors, comparison between bladder distension with lidocaine emda and distension with general or spinal anesthesia in the present study, there was significant improvement in both treatment arms at months 6 and 12 compared with baseline in terms of micturition frequency, mean voided volume, number of nocturia episodes, ic symptom and problem scores, vas score, and gra . Furthermore, this improvement in group b (hyaluronic acid instilled via emda) was significantly higher than that in group a (hyaluronic acid instilled via hydrophilic 12-fr foley catheter) for all parameters listed . This may mean that emda improves the efficacy of hyaluronic acid in patients with bps / ic as a result of increasing the tissue uptake of hyaluronic acid compared with that after installation with a hydrophilic catheter . Additionally, morales et al . Stated that the effectiveness of hyaluronic acid therapy decreased after 24 weeks . However, in our study group at 1 year, there was still significant improvement in all parameters with lower - dose hyaluronic acid than in the morales et al . This may relate to different groups of patients with different bps / ic pathogenesis . At month 24, the effectiveness of the therapy was decreased, and voiding frequency and vas were the only parameters that were significantly improved compared with baseline in both groups . At this point, the installation with emda group showed superiority to group a. the findings of the present study may suggest that emda improves hyaluronic acid efficacy; however, it does not increase the duration of efficacy . The number of patients in the study was low, and the findings should be confirmed in multicenter trails with a higher number of patients . Lack of a control group is a considerable limitation of the study; however, other studies of hyaluronic acid therapy have shown favorable findings in patients with bps / ic . On the other hand, we think that it is not ethical to treat this group of patients with placebo for a long time period . Plasma concentrations of hyaluronic acid could be determined in both groups; however, there were studies showing that emda increases the plasma concentration of the agent, so we did not perform this measurement . Hyaluronic acid installation is an effective gag substitution therapy in patients with bps / ic . Instilling hyaluronic acid via emda can improve the efficacy of the treatment; however, lack of long - term efficacy is the major problem of this gag substitution therapy.
Chromatin is made up of nucleosomes comprising histone octamers with a stable tetrameric core of histones h3 and h4, flanked by two more labile dimers of histone, h2a and h2b . Each histone octamer is wrapped by 147 bp dna, which facilitates the compaction of genomic dna and regulates access to regulatory factors (workman and kingston 1998). Chromatin is critical for the regulation of genome stability and for transcriptional control and its importance in disease has been highlighted by the frequent identification of mutations in chromatin - modifying enzymes in cancer genomes (plass et al . 2014). Intriguingly, sequencing of pediatric high - grade gliomas identified high - frequency mutations in a core histone subunit, h3 (schwartzentruber et al . 2012; wu et al . 2012), and subsequent studies have identified histone h3 to be mutated in virtually all cases of chondroblastoma and giant cell tumors of bone (behjati et al . 2013), diseases of adolescents and young adults . The majority of the mutations have been identified in genes encoding histone h3.3, which serves as a replacement histone as its deposition is not coupled to dna synthesis . Here, we review the specific characteristics of histone h3.3, the spectrum of mutations identified in tumors, and recent work directed at understanding how mutation of this protein contributes to disease . Several flavors of histone h3 are expressed in higher eukaryotes including histone h3.1, h3.2, h3.3, and a centromere - specific h3 variant protein, cenp - a . Histones h3.1 and 3.2 are synthesized during s phase (osley 1991), are incorporated de novo into newly replicated chromatin as well as during dna repair, and are thus termed dna synthesis - coupled . In contrast, the replacement histone h3.3 is expressed throughout the cell cycle, as well as in quiescent cells (wu et al . 1982), and is largely deposited in a dna synthesis - independent fashion by a distinct set of chaperones, proteins which associate with soluble histones and control the assembly (or disassembly) of nucleosomes from histones and dna . Histone h3.1 differs from h3.2 by a single amino acid (ser in h3.2), and h3.3 is distinguished by an additional four amino acid substitutions (ser, ala, ile, gly) (franklin and zweidler 1977) (fig . These clustered amino acids that differ between h3.1 and h3.3 have been linked to the differential binding of chaperones (tagami et al . 2010), with specifically g90 of h3.3 promoting binding to daxx (death - domain associated protein) (elsasser et al . 1histone h3.3 shows amino acid differences with h3.1 that promote binding to distinct chaperones . A sequence alignment of human h3.3, h3.2, and h3.1, with sequence differences in h3.3 marked in red . B cartoon of chromosome depicting regions of h3.3 incorporation into chromatin and the chaperones responsible histone h3.3 shows amino acid differences with h3.1 that promote binding to distinct chaperones . A sequence alignment of human h3.3, h3.2, and h3.1, with sequence differences in h3.3 marked in red . B cartoon of chromosome depicting regions of h3.3 incorporation into chromatin and the chaperones responsible the dna synthesis - coupled histones are encoded by unusual transcripts that lack introns and polyadenylation signals: histone h3.2 is encoded by three genes (hist2h3a, hist2h3c, and hist2h3d), whereas h3.1 is encoded by ten genes clustered on chromosome 6 . The dna synthesis - independent h3.3 is expressed from only two genes, h3f3a on chromosome 1 and h3f3b on chromosome 17 . These genes produce identical proteins even though they have distinct regulatory sequences and yield distinct polyadenylated transcripts with unusually long 5 and 3utrs (wells and kedes 1985; wells et al . The relative levels of h3.1 and h3.3 have been measured in several cell types and range from 2050% h3.3 and 2070% h3.1 in actively dividing cells (hake et al . 2006). However, given the cell cycle dependence of synthesis of h3.1 and h3.2, the relative abundance of h3 variants differs substantially between tissues and during development (gabrielli et al . Accordingly, post mitotic cells, such as cerebral cortical neurons, accumulate high levels of nucleosomal h3.3 (87% of nucleosomal h3 content) as dna synthesis - independent h3.3 deposition is ongoing while replication - coupled 3.1 and 3.2 deposition stops during gestation (pina and suau 1987). The dna synthesis - coupled and dna synthesis - independent h3s show distinct localization patterns across the genome . H3.1 is incorporated universally in the s phase by caf1 (chromatin assembly factor) (gaillard et al . 1996; tagami et al . 2004; ray - gallet et al . 2011) which is recruited to newly replicated dna by pcna (proliferating cell nuclear antigen) (shibahara and stillman 1999). Dna synthesis - independent h3.3 deposition occurs on dna sequences that are transiently nucleosome - free, for example, during transcription and dna repair, and to replace nucleosomes evicted by chromatin remodelers (filipescu et al . This seemingly contradictory pattern of h3.3 localization is due to the different chaperones that bind h3.3 (fig . 2010) in complex with the snf2-like remodeler atrx (-thalassaemia / mental retardation syndrome x - linked) whereas the hira chaperone inserts h3.3 into genic loci (goldberg et al . H3.3 accrues at actively transcribed regions (ahmad and henikoff 2002; schwartz and ahmad 2005; chow et al . 2005; jin and felsenfeld 2006) and can serve as a marker of regions of high transcriptional activity because it is preferentially deposited (over h3.1 and h3.2) in transcribed regions, and its histone tail is highly enriched for covalent modifications or marks associated with transcriptionally active chromatin, such as tri - methylation of lysine 4 (k4me3) (mckittrick et al . There is also conjecture over whether h3.3 is not just a marker for active regions, but whether it contributes to the transcriptional activity of loci enriched for this histone (huang and zhu 2014). This is in part due to its enrichment at regulatory regions such as promoters and enhancers, where it is thought to contribute to chromatin plasticity, or the openness of chromatin with enrichment for modifications associated with active chromatin and evidence of enhanced rates of nucleosome turnover and dynamic association of chromatin - associated proteins in these domains . Importantly, h3.3 is incorporated into polycomb response elements (pres) in drosophila (mito et al . 2007), regions of the genome that play critical roles in controlling gene expression during development, and is similarly enriched at promoter regions of developmentally regulated genes in embryonic stem cells (escs) by hira (goldberg et al . 2010; kraushaar et al . 2013 cells depleted for h3.3 show decreased levels of nucleosome turnover at sites of h3.3 incorporation (kraushaar et al . 2013; huang et al . 2013; banaszynski et al . 2013), correlating with defective expression of developmentally regulated genes on esc differentiation (banaszynski et al . H3.3 plays critical roles in stem cells, during fertilization and reproduction and during reprogramming of genomes following fertilization or somatic cell nuclear transfer (van der heijden et al . 2005; loppin et al . 2005; torres - padilla et al . 2006; hodl and basler 2009; sakai et al . 2009; santenard et al . 2010; banaszynski et al . 2013 h3.3 is also preferentially cleaved at residue 21 during senescence to lock in the senescent cell fate, presumably by removal of active mechanism to ensure nucleosome replacement in transcriptionally active areas (ray - gallet et al . H3.3 is incorporated at sites of uv damage, it protects against sensitivity to uv light and is required to maintain replication fork progression after uv damage (frey et al . H3.3 may also be important for the restart of transcription following dna damage since knockdown of the h3.3 chaperone hira resulted in an impaired recovery of rna synthesis after uvc damage (adam et al . Thus, h3.3 plays many diverse roles in chromatin regulation and is the subject of active study . Interest in h3.3 has increased even more with the surprising finding that the core histone h3 protein, and in particular, h3.3, is affected by specific mutations in several tumors . Histone h3 has recently been found to be mutated at high frequency in several specific cancer types including pediatric high - grade glioblastoma (hgg), chondroblastoma, and giant cell tumors of the bone (fig . 2). The identified missense mutations affect only three specific amino acids in the n - terminal tail of histone h3, a region of extensive posttranslational modification, and were found predominantly in the genes encoding h3.3, h3f3a, and h3f3b, and to a lesser extent in h3.1 genes, hist1h3b and hist1h3c . Such specificity and frequency of mutation allow these mutations to be defined as driver mutations for tumorigenesis (vogelstein et al . 2013). The specific mutations of h3.1 and h3.3 were found to vary by tumor type, patient age, and location, with each tumor containing a single mutant h3 allele (khuong - quang et al . 2specific histone h3 mutants arise in distinct regions of the brain or in different skeletal tissues and show variable age of presentation . A the amino acids that substitute glycine at amino acid 34 or lysine at amino acids 27 and 36 of histone h3, their properties, and possible posttranslational modifications . B cartoon depicting the different anatomical location of brain tumors bearing k27 m mutant h3.1 or h3.3 and g34r or g34v mutant h3.3 . K27 m mutants are predominantly found in midline structures (including the thalamus, pons, and brainstem), whereas the g34 mutant tumors are most commonly located in the cerebral hemispheres (sturm et al . C cartoon illustrating the distribution of different histone h3 mutants in chondroblastomas and giant cell tumors of bone (behjati et al . D graphical representation of span of age of presentation for histone h3 mutant tumors (schwartzentruber et al . 2012; sturm et al . 2012; behjati et al . 2013) specific histone h3 mutants arise in distinct regions of the brain or in different skeletal tissues and show variable age of presentation . A the amino acids that substitute glycine at amino acid 34 or lysine at amino acids 27 and 36 of histone h3, their properties, and possible posttranslational modifications . B cartoon depicting the different anatomical location of brain tumors bearing k27 m mutant h3.1 or h3.3 and g34r or g34v mutant h3.3 . K27 m mutants are predominantly found in midline structures (including the thalamus, pons, and brainstem), whereas the g34 mutant tumors are most commonly located in the cerebral hemispheres (sturm et al . C cartoon illustrating the distribution of different histone h3 mutants in chondroblastomas and giant cell tumors of bone (behjati et al . D graphical representation of span of age of presentation for histone h3 mutant tumors (schwartzentruber et al . 2012; sturm et al . 2012; behjati et al . 2013) sequencing of pediatric hgg tumors identified a recurring somatic mutation of h3 lysine 27 to methionine (k27 m) in 30% pediatric hgg tumors (wu et al . 2012), mainly in tumors of the midline such as the thalamus, basal ganglia, and spinal cord (sturm et al . Evidence for the mutation being somatic derives from sequencing of matched normal dna from patients carrying histone mutant tumors, which in every case (39 patients) demonstrated the somatic nature of the mutation (wu et al . The k27 m mutation is most often found in h3f3a (> 70%) with a few occurrences in hist1h3b (20%) and hist1h3c mainly in younger patients with a median age of 1011 years (wu et al . Analysis of the dna sequences for h3f3a and h3f3b illuminates why the k27 m mutation is restricted to h3f3a, as k27 is coded by aag in h3f3a and by aaa in h3f3b, requiring a single mutation in h3f3a to generate atg to code for methionine (fig . 3). Why the mutation has only been found in hist1h3b and c is less clear since six of the h3.1 genes have aag coding for k27 . This selection may be explained by differences in expression patterns of the different h3.1 encoding genes . Data from several groups indicate that diffuse intrinsic pontine glioma (dipg), a tumor of the pons, has an even higher incidence of k27 m, with> 70% of tumors sequenced containing the mutation as compared to less than 25% of non - brainstem gliomas (khuong - quang et al . Dipg tumors are particularly deadly, with a median age of onset of 8 years and survival rates of 10% at 2 years post - diagnosis (khuong - quang et al . 2012). Whether this poor outcome is linked to the preponderance of the k27 m mutation or to the inability to surgically resect these tumors can now be assessed using immunohistochemical staining of all hgg tissue samples with newly developed diagnostic anti - h3k27 m antibodies (venneti et al . Orange - shaded boxes mark the genes in which mutations are prevalent for the different amino acid substitutions codon usage in histone h3.3 and h3.1 genes at the sites of histone mutation . Orange - shaded boxes mark the genes in which mutations are prevalent for the different amino acid substitutions around 30% of non - brainstem pediatric glioblastoma tumors bear histone h3 mutations, including thalamic k27 m mutations . In cortical hggs, 15% bear a distinct mutation in histone h3.3, of glycine 34 to arginine (g34r) or, much less frequently, to valine (g34v). G34r / v containing tumors tend to be located in the cerebral hemisphere of the brain with nearly all g34r / v mutations found in h3f3a . G34 is coded by identical sequences in h3f3a and h3f3b, and r and v substitutions can be achieved with single point mutations . Similarly, g34r or v substitutions could be achieved by a single point mutation of any of the h3.1 genes, so other factors must determine the prevalence of h3f3a mutations in the tumors . G34 mutation is associated with global dna hypomethylation, which is particularly pronounced in telomeric regions (sturm et al . No g34r / v mutations were found in dipg tumors, and the median age of g34 mutant tumor occurrence was older than for k27 m mutant tumors (schwartzentruber et al . Additional sequence analysis of dipg tumors has highlighted the complexity of the genomic landscape, with identification of additional driver mutations that overlap or are excluded from tumors bearing mutant histones . In nearly 30% of glioblastoma tumors, the h3f3a k27 m mutation was observed in concert with mutations in atrx or less commonly in daxx (khuong - quang et al . Other studies show only a minor overlap between atrx mutation and k27 m (fontebasso et al . 2014) or no mutations in atrx in k27 m tumors (taylor et al . Also, k27 m tumors often contain mutations in the tumor suppressor protein p53, with nearly 60% harboring the mutation (khuong - quang et al . 2012; schwartzentruber et al . G34r h3f3a showed a significant overlap with mutations in atrx / daxx and p53 with nearly 100% of the tumors containing both mutations (schwartzentruber et al . Other somatic mutations that appear linked to h3k27 m mutation are activating mutations in acvr1 (activin receptor type 1) that enhance bmp (bone morphogenetic protein) signaling (fontebasso et al . Interestingly, mutation of acvr1 (which occurs in 24% dipgs) was linked to the presence of hist1h3b mutation (h3.1 k27 m) (buczkowicz et al . 2014; wu et al . 2014; fontebasso et al . 2014; taylor et al . 2014a) and was associated with a younger age of onset of disease (wu et al . 2014). Interestingly, while acvr1 mutations suffice to increase proliferation of immortalized normal human astrocytes (buczkowicz et al . 2014), mutations in the identical amino acids are found in the germline of individuals with the autosomal dominant congenital childhood disorder fop (fibrodysplasia ossificans progressiva), who have no evidence of cancer predisposition (jones and baker 2014; taylor et al . Thus, acvr1 mutation likely provides a selective advantage in the presence of other critical mutations, but cannot initiate tumorigenesis, as supported by the failure of p53 null mouse astrocytes that express acvr1 mutants to initiate tumorigenesis when implanted in the brain (wu et al . 2014). Idh1 mutations which are common in glioblastoma of young adults showed no overlap with h3f3a mutations (sturm et al . Inactivating mutations were also identified in the histone h3 k36 trimethyltransferase, setd2, in 15% of pediatric hgg . Setd2 mutations were initially thought to be restricted to cerebral hemisphere tumors and to show no overlap with h3f3a mutations (fontebasso et al . 2013), but setd2 has been found to be mutated in a dipg bearing a h3.1 k27 m mutation (wu et al . Mutations in h3.3 have also been found in several types of bone tumors, with the greatest incidence in chrondroblastoma and giant cell tumors of the bone (behjati et al . Chondroblastoma arises in children and in young adults in the cartilage of the growth plates of the long bones and is most typically benign . Sequencing of h3f3a and h3f3b in more than 70 chrondroblastomas revealed nearly 95% of the tumors contained lysine 36 to methionine (k36 m) substitutions, which mutate the target site for setd2 and other k36 methyltransferases . Unlike the k27 m mutation of glioblastoma, nearly all of the k36 m mutations were found in h3f3b (90%) rather than in h3f3a, which cannot be explained by differences in codon usage between h3f3a and h3f3b . Giant cell tumors of the bone also have a high frequency of h3.3 mutations, with greater than 90% of tumors sequenced containing substitutions of g34 to either tryptophan (g34w) or, in rare cases, leucine (g34l). A single base substitution suffices to mutate h3.3 g34 to w, whereas two or three mutations would be required to convert h3.1 g34 to w, and two mutations are required in any of the genes to generate a codon for leucine at position 34 . In contrast to the relative genetic complexity of the pediatric high - grade gliomas, the genome of these skeletal tumors is relatively stable, with cells being diploid and wild - type for p53 (behjati et al . This suggests that these h3.3 mutations may not only be defined as oncogenic drivers because of their high frequency of occurrence but may also be important drivers for effecting tumorigenesis in these tumors . Mutations of h3.3 were also observed at low frequency in osteosarcoma (2% containing g34r in h3f3a or h3f3b), conventional chondrosarcoma (1% containing k36 m in h3f3a), and clear cell chondrosarcoma (7% containing k36 m in h3f3b) (behjati et al . 2013). Interestingly, although highly prevalent in pediatric glioblastoma, to date, no k27 m mutations have been observed in bone or cartilage tumors, and the k36 m mutant has not been found in glioblastoma . The n - terminal tails of histones are decorated in covalent posttranslational modifications which can modulate the accessibility of the underlying dna for diverse chromatin transactions such as transcriptional control, chromosome segregation, and the repair of dna damage . 4a), and the k27 m missense mutation has generated much interest due to studies which suggest it plays a dominant role in blocking the accumulation of repressive h3k27 methyl marks (bender et al . 2013; lewis et al . 2013; venneti et al . 2013; chan et al . Pcg are evolutionarily conserved proteins which have roles ranging from seed development in plants, x - chromosome inactivation in mammals to maintenance of identity in stem cells . These complexes epigenetically regulate transcriptional states by modulating the h3k27me3 and h2ak119ub1 histone marks . In drosophila, pcg proteins interact with pres to establish cellular memory modules and are involved in developmental determination of body plan by repressing homeotic genes (reviewed in (di and helin 2013; grossniklaus and paro 2014)). While pre - like elements in mammals have been reported (sing et al . 2009; woo et al . H3k27 is an important target of prc2 (polycomb repressive complex 2) complexes that contain ezh2 or ezh1 histone methyl transferases . Indeed in drosophila, k27 has been demonstrated to be a critical target for prc2 since replacement of the histone h3 cluster with an h3k27r transgene mimicked the phenotype of loss of prc2 activity (pengelly et al . 2013), even in the context of a wild - type h3.3 genetic background.fig . 4k27 m mutants dominantly block prc2 methyltransferase activity on h3k27, whereas g34r / v mutants block setd2 methyltransferase function on k36 of the same tail . A representation of the amino terminal tail of histone h3.3 showing the position of known posttranslational modifications, the site of amino acid substitutions identified in tumors (k27 and g34: red), and the amino acid that differs in the h3.3 tail from h3.1 (ser 31: orange). B cartoon depicting the distinct modes of action of k27 m mutants and g34r / v mutants in modulating posttranslational modifications on h3 proteins . Note that for the k27 m mutant, we depict ezh2 as bound to mutant chromatin, with methyltransferase activity blocked on adjacent sites . Such binding of ezh2 on chromatin may block the chromatin template from additional chromatin transactions . An alternative possibility is that non - nucleosomal k27 m mutant h3 could sequester ezh2 off chromatin, which would leave open the possibility of additional modifications occurring on the mutant chromatin template k27 m mutants dominantly block prc2 methyltransferase activity on h3k27, whereas g34r / v mutants block setd2 methyltransferase function on k36 of the same tail . A representation of the amino terminal tail of histone h3.3 showing the position of known posttranslational modifications, the site of amino acid substitutions identified in tumors (k27 and g34: red), and the amino acid that differs in the h3.3 tail from h3.1 (ser 31: orange). B cartoon depicting the distinct modes of action of k27 m mutants and g34r / v mutants in modulating posttranslational modifications on h3 proteins . Note that for the k27 m mutant, we depict ezh2 as bound to mutant chromatin, with methyltransferase activity blocked on adjacent sites . Such binding of ezh2 on chromatin may block the chromatin template from additional chromatin transactions . An alternative possibility is that non - nucleosomal k27 m mutant h3 could sequester ezh2 off chromatin, which would leave open the possibility of additional modifications occurring on the mutant chromatin template detection of high - frequency k27 m mutations in pediatric dipg directed examination of the obvious link between h3k27- and prc2-mediated epigenetic modifications . K27 m mutation is predominantly found in h3.3, while 20% of k27 m mutations are found in h3.1 (wu et al . Most intriguingly, many studies have shown a dominant effect of the k27 m mutation, irrespective of whether found in h3.1 or h3.3, with a pronounced reduction in total h3k27 me2 and me3 in cells expressing one h3k27 m mutant allele among 30 alleles encoding h3 isoforms (lewis et al . Prc2, by stabilizing binding of the enzyme to k27 m and thus prevents deposition of methyl marks on other h3 proteins (lewis et al . K4 m mutants in h3 were previously used to mimic h3k4me2 and to stabilize binding of lsd2 to h3 for crystallization studies (zhang et al . The level of inhibition of prc2 activity by k27 m is similar to a known chemical ezh2 inhibitor, gsk343 (bender et al . Immunoprecipitation of k27 m co - purified ezh2 (chan et al . 2013) and the use of a photoreactive k27 m containing peptide to identify binding partners cross - linked primarily the ezh2 subunit of the prc2 complex (lewis et al . Substitution of k27 with methionine and to a lesser extent isoleucine seems sufficient to block the set catalytic domain of ezh2 by affecting substrate binding and turnover . A similar reduction in set domain activity was seen for k9 m and k36 m, but interestingly, not k4 m mutants, where lysines are targeted by distinct set domain containing methyltransferases (lewis et al . An interesting question is whether the stabilized binding of ezh2 to k27 m can occur on soluble histone h3 prior to its incorporation into nucleosomes, as suggested by in vitro binding studies to peptides (brown et al . If so, only ezh2 function would be blocked, whereas if ezh2 is bound to mutant chromatin, the activity of other nucleosome modifiers or chromatin remodelers may also be affected . Mis - regulation of prc2 target genes and mutations predicted to increase or reduce the activity of prc2 components have been reported in many cancers (hock 2012), but prc2 component mutations per se have not been found in glioblastoma . Mutation of p53 in conjunction with expression of h3k27 m in nestin expressing progenitor cells of the neonatal brainstem was not sufficient to induce glioma but did induce ectopic cell clusters in the majority of mice that stained positive for ki-67, which marks proliferating cells (lewis et al . 2013). The apparent induction of proliferation by expression of k27 m appears to be very cell type and developmental stage - specific as k27 m did not induce proliferation in undifferentiated human es cells or in primary human astrocytes (funato et al . 2014) and indeed suppressed the proliferation of immortalized normal human astrocytes (buczkowicz et al . 2014), suggesting that there is no driver effect on tumorigenesis of the k27 m mutation alone in this cell type . Sequencing of pediatric glioblastomas has revealed the presence of several other coexisting driver mutations in signaling pathway components and in other chromatin regulators in distinct classes of pediatric high - grade gliomas (reviewed in (jones and baker 2014)). Generation of mouse models will allow dissection of the interplay and contributions of histone h3 mutants and other mutations for gliomagenesis . At sites of high transcriptional activity, incorporation of h3.3 is increased relative to h3.1 and h3.2, so in cells expressing h3.3 k27 m, this may contribute to enhanced enrichment of the mutant protein in transcriptionally active domains . This may lead to a more potent effect of mutant h3.3 compared to mutant h3.1 in transcriptional dysregulation and contribute to tumorigenesis . In spite of the pronounced reduction in k27 methylation in gliomas expressing low levels of k27 m, chip - seq experiments have revealed that some genomic loci escape this effect and can accumulate high levels of k27me2/3 marks (chan et al . This enrichment for k27 methylation is associated with gene silencing, and genes in this group include cancer - associated genes such as p16ink4a and cdk6 . Genes that were reduced for h3k27me3 marks and were transcriptionally upregulated include the glioma - promoting candidate neural restricted transcription factor olig2 (chan et al . Additional modifications like reduction in dna methylation on oncogenic regions of the genome (bender et al . How these islands of prc2 activity are maintained is clearly an interesting question and may be linked to disparate modes of prc2 recruitment to different loci or to the use of alternate methyltransferases since to date there is no direct demonstration that ezh1 activity is similarly blocked by k27 m . Loss of h3k27me3 in k27 m expressing cells may also allow for an increase in h3k27ac (lewis et al . The presence of h3k27ac distinguishes active enhancers (calo and wysocka 2013; vermunt et al . 2014); thus, an aberrant transcriptional program may be elicited through turning on of normally silent enhancers in k27 m expressing cells, contributing to tumorigenesis . Gskj4, a pharmacological jmjd3 (a h3k27me3 demethylase) inhibitor, leads to restoration of h3k27me3, leading to tumor cell lethality in vitro and a significant improvement in the survival of mice that carry tumors (hashizume et al . Calls for alternative approaches by targeting different histone modification pathways to alter other posttranslational modifications on histones such as h3s28 phosphorylation which can minimize the dominant negative effect of h3k27 m recently it was found that co - expression of k27 m in the presence of other mutations (a constitutively active form of platelet - derived growth factor a and loss of p53) in neural progenitor cells derived from human embryonic stem cells promotes neoplastic transformation and induction of low - grade dipg (funato et al . Chemical screens on these induced dipg cells have identified the menin inhibitor mi2 as a potential drug candidate (funato et al . It is interesting to note that menin is a member of the trithorax histone methyltransferase complex and is involved in transcription regulation . Therefore, independent studies across models seem to point toward epigenetic pathways as potential therapeutic targets in treatment of pediatric hggs . However, the complexity of the genomic landscape of these tumors argues for the importance of performing biopsies to allow for better classification of individual tumors, and that targeting multiple driver mutations may be necessary to achieve therapeutic benefit . Lys 36 of histone h3 can undergo mono-, di-, and tri - methylation as well as antagonistic acetylation at the same residue . Posttranslational modification of h3k36 is associated with active transcription, alternative splicing, dosage compensation, dna replication, and dna damage repair (wagner and carpenter 2012). In yeast, set domain - containing 2 (set2) writes all three methylation states at h3k36 whereas in mammals, each state of methylation is laid down by distinct enzymes suggesting extensive regulation of k36 methylation and its importance throughout evolution (morris et al . 2005; wagner and carpenter 2012). For example, setd2 (the only enzyme that performs k36me2 to me3 modification) is mutated in renal carcinoma and breast cancer (duns et al . 2010; newbold and mokbel 2010), and nsd2, which generates k36me2, has been shown to be a tumor suppressor (kuo et al ., missense or truncating mutations in setd2 have been reported in pediatric high - grade gliomas of the cerebral hemispheres that do not harbor h3.3 mutations (fontebasso et al . 2013). It is presently unclear exactly how setd2 functions as a tumor suppressor, but loss of function may result in an impaired chromatin template for processes such as transcription and dna repair . Other tumors mimic loss of setd2 function by mutation of k36 of h3.3 (to k36 m) or by introduction of a mutation in a residue close to k36 (at g34) that may influence binding of writer or reader proteins at k36 (schwartzentruber et al . Why g34 is targeted, but not other neighboring residues of k36 remains a mystery . It is conceivable that the introduction of a charged (r) or a bulky residue (w) in place of g34 might impact the accessibility or activity of enzymes that target k36 or alter the conformation of the tail leading to changes in nucleosomal packaging that affect the binding of histone readers to h3k36me3 . Accordingly, nucleosomes harboring either a g34r or g34v mutant h3.3 exhibit reduced h3k36me2/me3 levels on the same tail, but have no dominant effect on total cellular h3k36me2/me3 levels (in contrast to the reduced k27me3 in h3k27 m mutants) (bjerke et al . 2013; lewis et al . It is intriguing that k36 to m and g34w / l mutations have been identified in h3.3 in chondroblastoma and giant cell tumors of bone (behjati et al . 2013), raising the important question of why the k36 methylation axis is targeted in cancers arising in developmentally distinct tissues . Also, it is important to note that the mutations impacting k36 methylation (k36 m and g34 mutants) are only in h3.3, with little rationale for this selection based on codon usage . The selection for these mutations in h3.3 may be explained by the enhanced deposition of h3.3 over h3.1 in transcriptionally active domains and regulatory regions . Deposition of mutant h3.3 would profoundly impact the transcriptional program since loss of k36 methylation negatively impacts transcriptional elongation (yoh et al . The most extensive analysis of the role of g34 mutants has been performed in a cell line generated from a pediatric glioblastoma harboring a g34v mutation in h3f3a . The g34v mutation was linked to an altered transcriptional status of the cells, with quite widespread changes in rna polymerase ii association and levels of k36 methylation when compared with a glioma cell line wild - type for h3.3 . One locus that was particularly induced was mycn, an oncogene implicated in pediatric glioblastoma (swartling et al . Transduction of g34v mutant h3.3 into normal human astrocytes or into transformed human fetal glial cells was sufficient to induce n - myc expression two- to threefold over cells transduced with wt h3.3 (bjerke et al . . Such reprogramming of transcription of oncogenes may be critical for tumorigenesis in g34 mutant tumors . Although global levels of k36 methylation appear unaffected, this mark is reduced on nucleosomes bearing g34r / v mutations, and there clearly are widespread changes in k36 methylation profile of the g34v glioma cell line analyzed (bjerke et al . 2013) (which may be indirectly caused by the altered transcriptional program of these cells). One possibility is that an altered local h3k36 methylation state may modulate the function of proteins that normally read the mark . One interesting example is zmynd11, a tumor suppressor protein which specifically reads h3k36me3 on the replacement histone h3.3 (guo et al . 2014c). G34v / r mutations compromise zmynd11 binding to the h3.3k36me3 peptide (wen et al . 2014c); however, a role for zmynd11 has not been shown to date in any of the pediatric glioblastoma models or in chondroblastoma or giant cell tumors of bone . It is likely that zmynd11 will be delocalized in chondroblastoma bearing h3.3 k36 m mutation, and its localization will be altered in other g34 mutant - expressing tumors where k36 methylation is locally reduced . It will be interesting to determine whether mutation of zmynd11 or setd2 occurs in these tumor types and if knockdown of these factors in the appropriate cell types recapitulates features of the diseases . A second possibility is based on the role of h3k36 in genome stability . Pediatric hgg is characterized by abundant somatic coding mutations, suggestive of defects in dna damage repair (jones and baker 2014). Indeed, microsatellite instability (msi) is very high in pediatric hgg (viana - pereira et al . Histones have been shown to be deposited in uvc - damaged regions to restore transcription (adam et al . 2013), and chicken bursal lymphoma dt40 cells either depleted for histone h3.3 or harboring h3.3 with g34r / v mutation are sensitive to uv (frey et al . 2014). This is not surprising since h3k36 methylation and setd2 are involved in dna damage repair (carvalho et al . 2014; jha and strahl 2014; pai et al . 2014; pfister et al . H3k36me3 may promote dna repair pathways such as mismatch repair since h3k36me3 recruits the mismatch recognition complex hmuts onto chromatin through association of the hmsh6 pwwp (pro - trp - trp - pro) domain with h3k36me3 (li et al . H3k36 modification (methylation or acetylation) may also impact the choice of dna repair pathway between nonhomologous end - joining (nhej) and homologous recombination (hr) (pai et al . 2014), and h3k36 methylation status has also been implicated in determining the timing of origin activity during dna replication (pryde et al . It is interesting that h3.3 is the predominant h3 used for chromatin repair in many cell types (adam et al . Further exploration of dna damage pathways in pediatric hgg, chondroblastoma, and giant cell tumors of bone should prove fruitful . The identification of high - frequency histone mutations in pediatric high - grade gliomas has given us an inroad to better understand and to treat these deadly tumors . Of particular interest is to determine how k27 m mutation in a single copy of histone h3 can have a dominant effect on global h3k27 methylation and, in particular, how some genes escape these effects and accumulate k27me3 . Is the mechanism of h3k27 m blockage of ezh2 activity restricted to ezh2 or does it impact ezh1 also? It is also critical to determine the mechanism by which g34 mutations influence setd2 function and how they and the k36 m mutation contribute to tumorigenesis . Is it linked to the nature of the replacement histone that can be incorporated into actively transcribed and regulatory regions in nonproliferating cells? Or is it through h3.3 incorporation into repetitive heterochromatic regions and linked to disruption of the genomic stability normally contributed by these specialized domains? Intriguingly, pediatric hgg mutant tumors are sometimes also mutated for atrx and or daxx (khuong - quang et al . 2012), which incorporate h3.3 into telomeres (drane et al . 2010; goldberg et al . Loss of atrx / daxx has been linked to lengthening of telomeres by a telomerase - independent process termed alt for alternate lengthening of telomeres (heaphy et al . Alt is associated with extensive genome rearrangements and defects in double - strand break repair (lovejoy et al . 2012), reviewed in (osullivan and almouzni 2014). Pediatric hgg bearing atrx or daxx mutations and mutant for h3.3 are characterized by alt (schwartzentruber et al . . It will be interesting to determine whether alt is a feature of histone mutant tumors that are wild - type for atrx / daxx, and whether skeletal tumors bearing distinct h3 mutations similarly display association with mutant atrx / daxx and alt . H3.3 is frequently associated with regions of transcriptional activity . In vitro, there is little difference in stability of nucleosomes bearing h3.3 in place of h3.1, but in vivo, h3.3 is frequently incorporated along with another histone variant, h2az, and these nucleosomes are inherently unstable (jin and felsenfeld 2007; chen et al . H3.3 has been documented to be important for determining chromatin plasticity of developmentally regulated genes in pluripotent mouse embryonic stem cells (banaszynski et al . The histone mutant tumors are likely caused by a defect in differentiation during development, and the presence of mutant h3.3 or aberrantly modified h3 proteins (due to dominant effects of k27 m or local effects of g34 mutant h3.3) at key regulatory elements may be critical to alteration of transcriptional programs leading to tumor initiation or progression . H3.3 is incorporated into promoters of developmentally regulated genes, and these loci bear both h3k27me3 and h3k4me3 marks, marking these promoters as bivalent domains poised for transcriptional induction (bernstein et al . H3.3 is required for establishment of the h3k27me3 mark in these domains (banaszynski et al . 2013), and it is easy to envisage that a switch to k27ac or possibly k36me3 may tip the balance to activation of aberrant developmental programs contributing to tumorigenesis . Much progress has been made in our understanding of these histone mutants since their discovery, but there remains much to be done to develop therapies for pediatric hgg and for the skeletal and bone tumors . We anticipate that the development of additional model systems to interrogate the function of mutant h3 proteins together with insight from analysis of tumors will fuel the collaborative synergy between experts in cancer biology, chromatin biologists, and chemical biologists to develop effective therapies for patients and to improve our understanding of the fascinating biology of histone h3.
Seroprevalence of hydatidosis is between 1.2 and 13.8% in the different region of iran (1). Brain involvement occurs in 12% of patients with hydatidosis and comprises 2% of all brain masses in the endemic region, especially among the children (2). Symptoms of the brain hydatid cyst vary by location and size of the cyst . In a retrospective study of 117 children in tunis, common symptoms were a headache, vomiting, hemiparesis, seizure, mood alteration, and skull deformity (3). More than 50% of patients with the brain hydatid cyst have multiple cysts in the brain, and about 18% have visceral involvement, which has 9% mortality rate (4). Surgical treatment and excision of the cyst ruptures of the cyst and anaphylactic reaction are the most frequent complication of the surgery with the prevalence of 10% . Here we present an adult male with a large brain hydatidosis and atypical symptoms, who was treated successfully by surgery . A 19 yr - old male livings in ardabil, northwest of iran, was referred to neurosurgery clinic in khanevadeh university hospital, tehran, iran for a headache and progressive left hemiparesis since a week ago . He was treating for depression and incuriosity for two months by a psychologist . During two weeks, he developed headache, vomiting, and in the last week, left - side paraparesis occurred . Peripheral neurological examination showed left hemiparesis with the force of three to fifth in all muscle groups and normal deep tendon reflexes . Brain contrast - enhancing magnetic resonance imaging (mri) showed a large round mass about eight centimeters in the right frontoparietal lobe and mildly shifting the cerebral hemisphere to the left, without any ring - enhancement or surrounding edema (fig . 1). Contrast - enhanced t1-weighted brain mri shows a large brain hydatic cyst in the right frontoparietal lobe with mildly shifting of the cerebral hemisphere laboratory results showed mild anemia (hemoglobin=13.1 milligrams per deciliter). Further laboratory and imaging findings were normal, except for antibody titer against echinococcosis, which raised fourfold above - normal range in igg . Oral treatment with albendazole at a dose of 400 mg twice daily was initiated, and the patient underwent surgical excision of the cyst . Right craniotomy through parietal bone with left side, head turning position was done under general anesthesia . A largely fluctuated mass with adhesion to surrounding tissue appeared after dural opening, and 3% warm saline infused around the mass during 30 min, which led to slowing delivery of the mass in the next 40 min (fig . 2). Surgical excision of the brain hydatic cyst via right craniotomy through parietal bone finally, free layers were sutured anatomically consequence, and the bone fixed by bone - adhesive glue . Oral treatment continued about three months . After one - yr follow - up, left hemiparesis improved, and the left side limbs forces raised to four - fifths . At this time brain mri cystic hydatid disease (chd) is an infectious parasitic disease, often caused by e. granulosus . This parasite distributes worldwide, and the main routes of transmission are ingestion of foods or water, which is contaminated by scolex or egg . 12% of the patients have cerebral involvement, especially in the parietal lobe (2). Brain hydatid cysts may grow up 110 cm a year and symptoms are often related to elevated intracranial pressure (icp) and focal neurological deficit (5). In our patient, symptoms of rising icp appeared in the advanced course of the disease and the main complaints, which resulted in seeking medication, were depressive mood alteration, personality instability, and apathy . Dowling technique is often used for surgical excision, which consists of craniotomy through a big opening hole, cortical dissection and hydrostatically delivery of the cyst using an injection of hypertonic saline (3), such as our patient . Ruptures of the cyst and anaphylactic reactions are the most common complication of the surgery, but other complications such as subdural effusion, hematoma, pneumocephalus or herniation must be considered (6). Emergent brain surgery was performed in a 13 yr - old male for cerebral herniation because of a massive brain hydatid cyst (5). Despite the significant size of the cyst and adhesion to surrounding tissue in our patient, he did not experience any complication . It seems that other related factors such as adhesion to surrounding tissue, the severity of intracranial pressure, access to the operative field, and expertness of the surgeon may influence the outcome . We believe that the best surgical technique for excision of the cyst is adequate craniotomy, gentle decortication and enough time spending during the surgery.
Medical files of the patients who were referred to the outpatient clinics of children s health and diseases of namik kemal university research, and application hospital were analyzed, and height, bodyweight, waist, and hip circumferences of the patients were recorded . Among these cases, patients who had undergone complete word counts because of medical requirement or routine control were included in the study, and their neutrophil, and lymphocyte parameters were evaluated . Medical files or the patients were screened, and those with viral - bacterial infection, insulin - dependent diabetes, congenital metabolic disease or hormonal disorder were excluded from the study group . A total of 130 obese (aged 615 years) and 57 (aged 715 years) healthy control subjects were included in the study . Age, and measured body parametres of the healthy control, and obese individuals participating in the study body mass indices (bmis) of the children were estimated based on bmi percentiles established by the centers for disease control 2000 (cdc) for children, and adolescents . Participants with bmis between 8595 percentiles were defined as overweight, and those over 95 percentile as obese . Blood samples to be analyzed for neutrophils, lymphocytes, and other hemogram parametres were placed in tubes containing k2 edta . Following 15 minutes of agitation, the samples were analyzed in pentra dx nexus (uk) device under room temperature . For biochemical measurements all biochemical tests were performed in cobas c 501 roche (japan) biochemical analyzer using commercial kits . In all analyses spss 17.0 (chicago, il .) Normality of continuous variables was checked with kruskal - wallis test . For comparison of data with non - normal distribution mann- whitney medical files of the patients who were referred to the outpatient clinics of children s health and diseases of namik kemal university research, and application hospital were analyzed, and height, bodyweight, waist, and hip circumferences of the patients were recorded . Among these cases, patients who had undergone complete word counts because of medical requirement or routine control were included in the study, and their neutrophil, and lymphocyte parameters were evaluated . Medical files or the patients were screened, and those with viral - bacterial infection, insulin - dependent diabetes, congenital metabolic disease or hormonal disorder were excluded from the study group . A total of 130 obese (aged 615 years) and 57 (aged 715 years) healthy control subjects were included in the study . Age, and measured body parametres of the healthy control, and obese individuals participating in the study body mass indices (bmis) of the children were estimated based on bmi percentiles established by the centers for disease control 2000 (cdc) for children, and adolescents . Participants with bmis between 8595 percentiles were defined as overweight, and those over 95 percentile as obese . Blood samples to be analyzed for neutrophils, lymphocytes, and other hemogram parametres were placed in tubes containing k2 edta . Following 15 minutes of agitation, the samples were analyzed in pentra dx nexus (uk) device under room temperature . For biochemical measurements all biochemical tests were performed in cobas c 501 roche (japan) biochemical analyzer using commercial kits . Normality of continuous variables was checked with kruskal - wallis test . For comparison of data with non - normal distribution mann- whitney measured parametres of a total of 187 healthy, and obese children are given as median, minimum, and maximum values in table 2 . Obese, and healthy control groups were compared, and lymphocyte, and neutrophil ratios were found to be significantly higher in the obese group (p=0.03, and 0.045, respectively). Similarly, neutrophil / lymphocyte ratio, and crp levels were found to be significantly higher in the obese group (p=0.03, p=0.02, and p=0.00). Besides, mean platelet volume (mpv) was significantly higher in the obese group (p=0.005). Both groups did not differ significantly as for platelet / lymphocyte ratio (p=0.156) biochemical, and hematological parametres measured in healthy controls, and obese individuals p<0.05 statistically significant; hgb: hemoglobin; hct: hematocrit; mcv: mean corpuscular volume; mch: mean corpuscular hemoglobin; mchc: mean corpuscular hemoglobin concentration; rdw: red blood cell distribution width; wbc: white blood cell; mpv: mean platelet volume; pdw: platelet distribution width; crp: c - reactive protein . In recent years because of diets rich in fats, and sedentary life style, prevalence of obesity has rapidly increased . Obesity plays a central role in insulin resistance which include hyperinsulinemia, hypertension, hyperlipidemia, and type-2 diabetes, and increases the risk of cardiovascular disease . Although, obesity has been demonstrated to be an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism of increased cardiovascular risk in obese individuals has not been clarified yet . In studies performed, the pathogenesis of pathological processes of atherosclerotic cardiovascular disease, and related risk factors have been dated back to the pediatric age . Obesity is a chronic inflamatory disease characterized by altered adipokine production, and increase in the levels of inflammatory cytokines . Kim et al . Analyzed levels of leptin, and inflammatory cytokines including hs - crp, tumor - necrosis factor (tnt-), interleukin-6 (il-6) in young obese girls, and they were found to be significantly higher in obese women relative to non - obese women . In recent studies increased levels of crp were detected in obese individuals [14, 15]. High - sensitive c - reactive protein (hs - crp) which can be used as a marker of metabolic syndrome is a useful marker of childhood obesity . Prevention of obesity, and inflammation which start in childhood will contribute to the prevention of metabolic syndrome in the future . Similarly, crp level in the obese group was found to be significantly higher in our study when compared with the healthy controls . Arslan and makay have demonstrated that mpv levels were higher in obese adolescents with non - alcoholic fatty liver when compared with healthy controls, and other obese patient groups . However, kilciler et al . Reported that any significant difference was not found between the patients with non - alcoholic fatty liver disease as for mpv levels . Also in our study, we detected higher mpv levels in the obese patient group when compared with the healthy controls . Nlr which is a strong indicator of inflammation has been investigated in various inflammatory, and neoplasic diseases including metabolic syndrome, behcet s disease, coronary artery disease [21, 22], heart failure [23, 24], colorectal cancer, chest, and lung diseases, and hepatocellular carcinoma . The main fundamental etiological factor of this wide spectrum is that nlr contains two separate inflammation markers . Neutrophils have been reported to migrate into ischemic myocardial tissue before other inflammatory cells, and cause destructive changes by stimulating release of proteolytic enzymes, reactive oxygen radicals, and neutrophils . As a known fact, in the pathophysiology of atherosclerotic cardiovascular diseases, increased inflammation, and endothelial dysfunction play fundamental roles . Outcomes of this study suggest that nlr may be one of the markers of endothelial dysfunction, and inflammation in obese patients . Related studies performed in adults have suggested that various comorbidities as diabetes, insulin resistance, hyperlipidemia, and coronary artery disease prevent disclosure of cause - effect relationship between obesity, and inflammation . Therefore this study provides a clarification on this issue which also aids in the evaluation of this correlation between obesity, and inflammation . In conclusion, in chidhood obesity, therefore keeping obesity under control using dietary modifications, and other treatment methods carries importance in decreasing mortality, and morbidity rates in adulthood.
Recent technological advances in the field of radiation oncology are revolutionizing the management of cancer with ionizing radiation . Through the use of highly conformal techniques, the ability to deliver curative doses to sub - millimeter accuracy is unprecedented to now . In particular, intensity - modulated radiotherapy (imrt) has had a substantial impact on the management of head and neck carcinoma (hnc), and its use is highly prevalent among radiation oncologists . Imrt allows for the delivery of high doses to target volumes while simultaneously limiting the dose to organs at risk, so that once common toxicities, such as xerostomia, can be limited . However, for this to be achieved, sharp gradients in dose are produced, and therefore small changes in patient or tumor position may have large dosimetric implications . In particular, several studies have demonstrated that patient / tumor motion during imrt specifically for hnc is clinically significant [24]. Image - guided radiotherapy (igrt) is a novel array of techniques to help minimize the discrepancies due to variations in patient / tumor position . A strict definition of igrt is the use of images to monitor or modify treatment delivery . However, igrt can also be divided into three broad categories of image - based innovations: (1) the integration of functional and biological imaging into the treatment planning process to improve tumor contouring (or target delineation), (2) the use of various imaging modalities to adjust for tumor motion and positional uncertainty, and finally (3) the adaptation of treatment planning based on tumor response and changes in normal tissue anatomy . The latter form of igrt, known as adaptive radiotherapy, has the potential benefit of avoiding unintended normal tissue toxicity by altering the original treatment plan according to changes that may have occurred during the course of radiotherapy . Treating hnc is often complex, owing to the importance of preserving critical organ functions, such as salivation, speech, and swallowing, that are key factors in determining quality of life after treatment . Since radiotherapy continues to play a central role in the definitive [610], adjuvant [11, 12], and recurrent disease settings of hnc, it is likely that these innovations will continue to improve outcomes by minimizing toxicity and maximizing organ preservation . In addition, dose escalation with imrt may lead to improved local control, which may ultimately extend survival if augmented by improvements in systemic therapies for metastatic disease . Although many of these sophisticated imaging and treatment modalities that employ igrt are still yet to be proven beneficial in randomized controlled trials, the theoretical benefits of improved disease - control and normal tissue sparing are currently being demonstrated in a variety of peer - reviewed publications, which is the focus of the following review . The first type of igrt involves the incorporation of new diagnostic imaging modalities into the initial tumor contouring stage of radiotherapy planning in order to more precisely identify areas that should be treated with radiation . Currently, most centers employ ct - based planning, where the patient is simulated in the treatment position and then the targeting of macroscopic and microscopic disease sites is performed on ct - acquired images alone . Although ct - based planning is common for hnc, recent studies have suggested that a large degree of interobserver variability exists in the contouring of the gross - target volume (gtv). Cooper et al . Asked eight expert physicians to contour the same gtv in 20 patients with supraglottic carcinomas and found that the overlap in contoured volumes was only 53% with ct - alone . As precise tumor localization is of growing importance with increasingly conformal radiotherapies, attention has now shifted to novel forms of imaging that provide additional biological and tumor information that can be included in the planning process in order to clarify areas of tumor burden . A key innovation in this form of igrt is the use of 18-f - flurodeoxyglucose (fdg)-pet . Fdg is a radiolabeled analog of glucose that is selectively absorbed in tumor cells more than normal tissues, and thus it is useful in distinguishing neoplastic growth in tissues that otherwise appear radiographically normal . As such, fdg - pet has a well - established role in oncology and is commonly used in tumor staging for several cancers, including hnc [1517]. However, increased interest has now focused on the use of fdg - pet in target delineation for radiation therapy in order to guide the contouring of tumor margins and extended fields . Since most tumor contouring is performed on ct - based images, this is accomplished by using sophisticated software to perform an accurate overlay (or registration) of pet and ct images . In this alternatively, some centers are now equipped with hybrid pet - ct scanners that are capable of acquiring both pet and ct scans during a single session . This has the added benefit of imaging the patient while in the treatment position . Research on fdg - pet in hnc has shown that pet - based planning can significantly influence the size of the gross - tumor volume (gtv) that is outlined [1924], the size of the nodal volume [23, 24], and assist in the detection of nodal metastases not visualized or enlarged by ct criteria [23, 24]. Most studies have found that pet - based planning tends to reduce the gtv, however some studies have shown that pet - based planning can also increase the size of volumes contoured [5, 19]. Furthermore, new clinical evidence from patients treated with pet - ct planning is appearing in literature . Research has shown that pet - ct based planning can lead to excellent local control [18, 25], significant alterations in staging, and decreased normal tissue toxicity . In particular, vernon et al . Reported on 42 patients with hnc who underwent pet - ct during planning and were followed for a median of 32 months . A high level of disease control was obtained, and acute toxicities were relatively mild and improved with time . Although the initial results of improved tumor localization through pet - ct planning are optimistic, several areas of concern exist . Raise an issue regarding pet - ct planning in a recent study of 38 patients who were planned using pet - ct and ct - alone . These researchers found that although the gtv was reduced in 92% of patients with the addition pet - ct from ct - only - based plans, the planning target volume (which includes areas of microscopic disease and additional margins for error) was not significantly different between the two planning modalities . As such, no clinical advantage would be expected from the combined pet - ct planning . Further research on technical issues such as this will have to be carefully addressed in the future before widespread implementation of these technologies . As of now, fdg - pet has a well - established role for tumor staging, monitoring tumor response, and follow - up of hnc patients . However, the routine use of pet - ct for planning is not yet recommended . Fdg - pet is a commonly used radioactive tracer; however several novel tracers are being employed in hnc imaging . Tumor hypoxia is a common occurrence in the tumor microenvironment and has a well - known role in the resistance of tumors to radiotherapy . Furthermore, it is thought that many hypoxia - induced treatment failures can be prevented in part by escalating the dose to hypoxic subvolumes of the gtv . However, this process depends on our ability to accurately identify hypoxic areas and deliver a targeted radiation boost to those localities . [f]-misonidazole (fmiso) is a novel tracer that has been shown to accurately identify hypoxic areas in head and neck tumors [2830]. Have used fmiso - pet to identify hypoxic subvolumes in 10 hnc patients and subsequently escalated the dose to those areas with a local boost . No outcomes were reported, but the feasibility of the technique has been established . A recent study describes the treatment of 20 hnc patients who received routine pre- and mid - treatment fmiso scans in order to determine the effect of tumor hypoxia on patient prognosis . Surprisingly, these results showed that neither the presence nor absence of tumor hypoxia as defined by fmiso was correlated with patient outcome . Although this may suggest that tumor hypoxia is not correlated with patient outcomes, the authors suggest several alternative explanations to this idea, including the notion of tumor reoxygenation during fractionated radiotherapy . Furthermore, a wealth of preclinical and clinical data support the worsening prognosis associated with hypoxia in hnc [27, 3336]. In any case, further investigation is necessary to ascertain whether the outcomes of hnc can be improved by specifically targeting hypoxic zones . Other non - fdg tracers have also been investigated for their role in hnc patients . In particular, 1-(c)-acetate pet (ace - pet) has been shown to be a promising tracer for hnc staging and target delineation and may be used to complement fdg - pet . The molecule, 3-deoxy-3-f - fluorothymidine (f - flt), is also of growing interest to hnc management . Flt is phosphorylated by the cytosolic enzyme thymidine kinase-1 (tk1) and is subsequently trapped intracellularly . Tk1 activity is increased during dna synthesis, and thus f - flt trapping is a marker of proliferation . Research specifically in hnc has shown that flt uptake is correlated with decreased survival, has good reproducibility and may potentially be useful in determining tumor response to radiotherapy . Finally, similar to fmiso, cu(ii)-diacetyl - bis(n(4)-methylthiosemicarbazone) (cu - atsm) is a marker of hypoxia but through an entirely different mechanism . This tracer has also been evaluated in hnc and was shown to provide another feasible approach for hypoxia - directed radiotherapy . However, further research is necessary before the routine implementation of this or other novel pet tracers into daily clinical use . The second type of igrt involves the use of modern imaging modalities to assist in daily patient positioning . Most radiotherapy protocols involve several weeks of sequential daily treatment, and each day the patient needs to be repositioned into the exact position obtained during the initial planning ct . However, often small positioning errors occur, which introduces the possibility for considerable interfraction motion . In addition, if the patient is not properly immobilized during a radiotherapy session, there is also the potential for intrafraction motion . As six potential degrees of freedom are prone to changing between and during fractions, accurate positioning is an exceedingly complex challenge . Over the years, several unique methods have been devised to address and minimize interfraction and intrafraction motion . Traditionally in hnc, thermoplastic masks composed of a mesh - like grating are placed over a patient's face and secured to the treatment couch in order to immobilize the patient's head during ct - simulation and treatment . The masks have markings on them that allow the radiation therapists to then re - position the patient prior to each fraction with the aid of optical arrays . Various types of masks [3, 4347] and bite blocks have been employed for hnc patients . However, due to the flexibility of the head and neck region, these immobilization techniques have a potential for considerable setup variability [45, 4951]. Another common way to verify patient positioning is through the use of two - dimensional (2d) portal film imaging (see figure 1). This is done using devices attached to the treatment machine that are capable of taking two - dimensional megavoltage (mv) [48, 52, 53] or kilovoltage (kv) radiographs . Typically, this is performed at the beginning of each week of radiotherapy; however newer schemes have been devised for daily kv imaging that are more sensitive to day - to - day interfractional changes . Although these 2d - radiographs are adequate for detecting large positioning errors, they are problematic for a number of reasons . First, they tend to have poor image quality, making it difficult to identify set up inaccuracies [5658]. Second, they can only visualize bony structures, so changes in soft tissue are not detected using this method . Third, 2d - radiographs are not adequate for detecting rotational movement of the head [49, 59, 60]. As such, recent advances in three - dimensional (3d) (or volumetric) in - room imaging have offered new solutions to the limitations of conventional patient positioning . One solution that has been proposed involves the use of a conventional ct scanner mounted on a rail system, which is placed in the treatment room and shares a couch with the linear accelerator . This system is capable of taking high - quality, three - dimensional images after patient immobilization in order to verify setup between day - to - day treatments [51, 61, 62]. These images are of higher quality than traditional portal images, and they provide adequate resolution for soft tissue identification . However, the ct - on - rails system does have some distinct disadvantages . First, the addition of a full - size ct gantry into the treatment room can be cumbersome . Second, these systems are incapable of detecting intrafractional motion . Finally, this system introduces the need for movement of the couch between the ct scanner and the linear accelerator, which increases the time of the procedure . Cone - beam ct (cbct) is another novel form of 3d in - room imaging that can minimize patient positioning inaccuracies . Cbct is a scaled - down version of a ct scanner that is built into the treatment machine . Images taken from a cbct at the time of treatment can be overlaid on the original planning ct, and specialized software can be used to detect positioning errors with millimeter accuracy . Similar to 2d portal imaging, two types of cbct exist: mv and kv . Cbct with kv imaging is reported to have better image contrast and smaller signal - to - noise ratios than mv cbcts . Cbct imaging has been used to correct for interfractional motion in a clinically feasible amount of time . In addition, this technology is being studied for the detection of intrafractional motion, which could potentially be used for improved accuracy as well [65, 66]. Finally, cbct - based correction has also been used to increase treatment accuracy in the setting of imrt, thus allowing for larger target doses, while simultaneously sparing healthy tissues [67, 68]. There are concerns; however, about the additional radiation dose delivered with frequent cbct imaging [69, 70]. In particular, studies have estimated that daily cone - beam ct imaging can lead to an increase of 5.36.7 cgy to skin per scan and a total of 300 cgy over an entire treatment course . No long term data on the actual incidence of secondary malignancies is currently available, and continued investigation will have to be performed to address this question . In the past few years, helical tomotheraphy (ht) has become an increasingly popular technique that employs daily volumetric imaging to visualize both patient setup errors and tumor / organ variations [73, 74]. Ht combines a 6 mv ct with a therapeutic linear accelerator that is mounted onto a ring gantry . During treatment, the patient is translated through the ring while the gantry continuously rotates, resulting in helical fan beam radiation delivery . The radiation beam is dynamically modifed using a binary multileaf collimator, which allows for imrt and the creation of highly conformal dose distributions . In addition, using the on - board 6 mv ct scanner, daily image guidance can be performed with the patient in the actual treatment position . Research on ht in hnc patients has been promising . A prospective evaluation comparing ht to 3d - conformal radiotherapy (3d - crt) in 60 patients with disease at various anatomic sites found that ht was subjectively equivalent or superior to 3d - crt in 95% of plans . Furthermore, studies have shown that ht can achieve sharper dose gradients, improve dose homogeneity, and provide better sparing of the parotids than conventional imrt [7779] or stereotactic radiosurgery . Ht with daily position corrections using mv ct is also safe and easy to implement into a daily clinical routine . Clinical outcomes using ht in hnc patients have also been encouraging and have shown decreased dose, as well as toxicity, to the parotids without compromising survival, locoregional control, and disease - free survival in comparison to conventional and non - ht imrt approaches [81, 82]. Digital tomosynthesis (dts) offers another method of 3d in - room imaging for patient setup verification . Similar to cbct and ht, dts provides volumetric tomographic imaging; however it works by reconstructing 3d slices from a limited number of 2d cone - beam projections . These images may be of a lower resolution; however advocates of this technology argue that it is comparable to cbct in terms of imaging quality . Furthermore, since dts constructs images from a limited number of arcs, it may result in lower cumulative doses, as well as reduced treatment times in comparison to other modalities [83, 84]. These advantages may be of added benefit to pediatric patients, where reduced dose and decreased treatment times are a high priority . Several groups have reported on systems utilizing in - room cameras for imaging 3d surface reconstructions in real time [8688]. Others have used specialized cameras with infrared markers for determining target position [3, 8993]. These systems are reported to detect setup errors with high precision, as well as little setup time . This technology has also been used in combination with in - room radiographic imaging with promising results [44, 9496]. Unlike other radiographic modalities, optical modalities are noninvasive and do not expose the patient to added radiation dose . In addition, these techniques account for intrafraction motion and can be done in a relatively short amount of time . In conclusion, volumetric (3d) imaging in the hnc setting is superior to conventional 2d portal imaging in many ways . However, the extent to which this technology should be applied is unclear . In particular, the frequency with which 3d imaging for setup verification should be performed is unknown and is the subject of current debate . Some have argued that weekly or biweekly scans are adequate, while others have suggested that daily scanning is necessary . Art is a new, and still evolving, concept with the potential to greatly improve the delivery of radiotherapy . The current standard of treatment planning in radiotherapy involves obtaining an image at the start of treatment . The plan is then generated on that image and delivered to the patient over the course of his / her therapy . We know in head and neck cancer; however, that over the course of the 67 weeks of radiotherapy, there can be significant changes in the patient's anatomy based on shrinkage of the primary tumor or involved lymph nodes and loss of overall body weight [98100]. Applying the original plan to the now altered patient anatomy can lead to increasing the dose delivered to the surrounding normal tissues, including the parotid glands and spinal cord [101104]. Sparing these normal tissues is an important consideration, because post - radiation xerostomia has a significant impact on quality of life [105107] and dose constraints regarding the spinal cord and brain stem are always of concern due to potentially devastating consequences . Art allows us to adapt the treatment plan in response to the changes that occur so that we can maximally spare these normal tissues while maintaining complete coverage of the tumor volume . A study by barker et al . Examined the rate of tumor regression and the total overall tumor regression by obtaining ct images during treatment 3 times per week over the course of radiotherapy and quantifying the volumetric and geometric changes that occurred . They estimated that the gtv decreased by a median rate of approximately 1.8% per day . The median total gtv decrease was approximately 70% (range 10%92%) over the course of treatment, and this shrinkage tended to be asymmetric . The parotid volume also decreased by a median of 28.1% and moved medially with a median translation of 3.1 mm which correlated with patient weight loss . Demonstrated a median reduction of parotid volume of 49.8% and a median medial translation of the parotids of 8.1 mm over the course of treatment . Medial translation of the parotid glands from tumor regression and patient weight loss tend to bring the parotids into higher dose regions and therefore increase the dose to the parotids . In addition, shrinkage of the parotids can result in a much larger percentage of the parotid receiving high doses than anticipated . Estimated that the median dose increase to the ipsilateral parotid was 3 gy, and 45% of patients experienced increases between 57 gy . Though these doses seem small, the parotid is a very radiosensitive tissue and even small changes in dose can have a large impact . Blanco et al . Estimated that salivary function decreased at a rate of 5% per 1 gy increase in mean dose . They also noted that 70% of patients that received a mean dose of greater than 26 gy to both parotids experienced grade 4 xerostomia . In order to avoid the unintentional overdosing of the surrounding tissues, some investigators have studied re - planning the radiation treatments in response to changes in patient anatomy . Kuo et al . Performed a prospective trial in which 10 patients with enlarged lymph nodes were re - planned after delivery of 45 gy . Twenty - one gy was then delivered according to the new plan to complete the radiation treatment . The patients were then analyzed to compare the differences between the dose that was delivered after re - planning to the dose that would have been delivered without re - planning . Their results show that the dose to the parotid glands was reduced by approximately 24 gy by re - planning . Hansen et al . Performed a retrospective analysis on patients that were re - planned for weight loss or tumor regression . Comparison of the two plans showed that not re - planning led to decreases in target coverage and increases in dose delivered to the surrounding tissues . They found that the dose to 95% of the planned target volume was reduced in 92% of patients in the old plan compared to the new plan (range, 0.27.4 gy). In addition, the maximum dose to the spinal cord was higher in the original plan compared to the new plan in all patients (range 0.2 to 15.4 gy). The brainstem maximum dose was also increased in 85% of patients (range 0.68.1 gy). Though research in the field of art is mostly preliminary, it does show promising evidence of an improvement in the delivery of radiotherapy . Though the theoretical benefits of art are highly desirable, there are still many barriers to overcome before widespread adoption will be feasible . First, it is unclear when and how often re - planning should be done . Would re - planning once be sufficient or would it need to be done more frequently, such as weekly or even daily? Alternately would it be more appropriate to develop defined thresholds that, if met, would necessitate re - planning? Attempts are underway to identify the optimal re - planning schedule, but for now, this schedule must take into account the technical difficulties and the time required to create a new plan . Currently, occasional re - planning can be done, but frequent re - planning would overwhelm the available resources . New technologies such as deformable image registration and automated target delineation in conjunction with higher computational power will be required before widespread adoption of this new strategy can occur . In the future, igrt will likely continue to expand by incorporating newer and more sophisticated imaging modalities . In this section, we briefly discuss several cutting - edge technologies that are in the early stages of investigation in hnc, including molecular - based ct, high - resolution ultrasound, magnetic resonance imaging (mri), and proton therapy . Molecular - based ct imaging is a promising modality that may offer several advantages for tumor delineation . As ct is one of the most commonly employed diagnostic imaging modalities in hospitals today, it has widespread availability and convenience of use . However, ct is generally not thought of as a molecular / cellular imaging modality owing to the lack of targeted contrast agents . A recent report by popovtzer et al . At the university of michigan at ann arbor has described the use of gold nanoparticles that selectively and sensitively target tumor antigens . Using in vitro models of hnc, these researchers demonstrated that the attenuation coefficient for molecularly targeted cells is over 5 times greater than for normal cells . As such, nanotechnology - based ct may improve target delineation by providing more accurate microtumor identification during planning . Furthermore, since ct is easily accessible to most physicians, this technique could be rapidly introduced if proven to be both feasible and efficacious . Aside from ct and pet, several other imaging modalities have also been investigated for their potential role in radiotherapy planning for hnc . High - resolution ultrasound was studied by wein et al . Who demonstrated a feasible method for incorporating ultrasound - based information of the architecture of cervical lymph nodes into the planning ct for target delineation . Mri has also been examined in hnc . A recent study by gardner et al . Has found that mri fused to the planning ct can decrease the amount of interobserver variation in critical organ and target volume delineation for patients who have intracranial tumor extension, heavy dental work, or contraindication for contrast - enhanced ct . To these authors knowledge, no clinical data has yet been reported . However, based on these preclinical studies, mri and high - resolution ultrasound may contribute to improved outcomes in hnc patients . Proton therapy is another appealing form of radiotherapy owing to its superior dose distribution properties, which allow smaller volumes of normal tissue to be irradiated than is possible for any photon beam technique . Accordingly, initial clinical experiences of proton therapy in hnc have been encouraging and have shown reduced normal tissue toxicity in sinonasal, nasopharyngeal, and oropharyngeal malignancies . Although long - term efficacy studies are still immature, the preliminary data is encouraging . Furthermore, recent interest in combining proton therapy with modern improvements in image - guidance and dose - localization has arisen . In particular, just as the intensity of photon beams can be modulated in imrt, the intensity of proton beams can also be modified to produce intensity - modulated proton therapy (impt). Although a mature technique is still unavailable, an offline study in hnc patients has shown that impt has a better ability to spare organs at risk and is associated with a significantly reduced risk of secondary malignancy induction in comparison to imrt with photon beams . The feasibility of combining proton therapy with various forms of igrt, such as mri- and kv - based modalities, has also been demonstrated and may lead to a further reduction in normal tissue toxicity when clinical data becomes available [115, 116]. Based on preliminary reports such as this, future proton - therapy research is eagerly anticipated . With the advent of highly precise conformal therapies, such as imrt, the accurate localization and delivery of radiotherapy will be increasingly important in the decades to come . Recent advances in image - guided radiotherapy provide increased tumor localization by improving the identification of areas of tumor burden, by minimizing the effects of patient setup errors caused by intra-/interfraction motion, and by allowing for adaptive replanning to changes that occur in the tumor or patient during long courses of radiotherapy . In doing so, these changes are leading to improvements in the therapeutic ratio, where doses are increased at diseased - sites and minimized at normal tissues . Importantly, there are large financial and educational barriers in the initial setup and implementation of new imaging modalities . Furthermore, there is still no existing level i evidence demonstrating the benefit of igrt over standard radiotherapeutic modalities . Evidence from existing retrospective and nonrandomized studies; however, strongly supports the beneficial role of igrt . Further research is currently under way, and the results are expected to continue to validate the use of igrt in the management of hnc patients.
Padma 28 is a multicomponent, traditional tibetan herbal plant remedy comprised of 20 specific herbs and 2 nonherbal ingredients . The main padma's active substances are bioflavonoids, tannins, phenolic acids, phenolic alcohols, and terpenoids [1, 2]. Both aqueous and alcohol - based padma 28 preparations exhibited evident antibacterial effects against gram - positive bacteria and klebsiella pneumonia in vitro . From 20 herbs present in padma, 13 have well - documented antimicrobial activity . This herb also presents anti - inflammatory and immunotropic activity, enhancing th2 and suppressing th1 cytokine release by lymphocytes . Recently, chemopreventive effects of cardamom on chemically induced skin carcinogenesis in mice were described . Sida cordifolia, known for its regenerating properties, is active against corynebacterium diphtheria and in combination with nystatin and clotrimazole exhibited antimicrobial effects against five candida strains . Terpenoid eugenol present in syzygium aromaticum expresses general antimicrobial effect; phenolic alcohol from glycyrrhiza glabra was active against mycobacterium tuberculosis, staphylococcus aureus, and plasmodium [2, 49]. Recently, anti - influenza viral effects of nuclear export inhibitors from valerianae radix were described . Another padma 28 component, saussurea lappa root, and its active principle dehydrocostus lactone inhibit prostate cancer cell migration in vitro and have been shown to have anticancer activity . Santamarin, a sesquiterpene lactone isolated from this herb, represses lps - induced inflammatory response in murine macrophages and potently inhibits the growth of trypanosoma brucei rhodesiense [1115]. The plant possesses multiple activities, among them, antioxidant, antimicrobial, anti - inflammatory, and wound healing activities . Some immunotropic activities of padma 28 and beneficial effect of this remedy in experimental models of inflammation and wound healing were reported [1724]. In humans, padma 28 has been used as a beneficial tonic for heart and blood vessels and as an antioxidant . Padma 28 has been registered in switzerland since 1977 by intercantonal office for the control of medicines as a remedy to alleviate symptoms of claudication, impaired peripheral circulation, pain on walking, leg cramps, and paresthesia . A profitable influence of padma 28 was also observed in patients with atherosclerosis and in patients with multiple sclerosis [25, 26]. In 1992, its efficacy was further proved in prophylactics and treatment of some disorders with inflammatory, sclerotic, and degenerative origins . Treatment of chronic infective pulmonary diseases studied in poland in a big group of children with padma has brought positive results [27, 28]. However, as padma is being used for a variety of diseases and usually for a long time (e.g., couple of weeks) and because it possesses strong antioxidative properties, it would affect various parameters of immune system, among them, oxidative burst of granulocytes . In fact, some authors reported that this remedy inhibited the respiratory burst of human neutrophils in vitro [29, 30]. That is why we decided to evaluate in the present study, on the experimental model in mice, the in vivo effect of padma 28 (in high dose, comparable to that recommended for humans, and in low dose, comparable to these which we used previously in studies of various other herbal extracts) on immunological angiogenesis and, simultaneously, on granulocytes metabolic activity evaluated by chemiluminescence . This test is widely accepted as a method of measuring granulocytes oxygen - dependent killing potential . It is important to know if selected doses of this remedy do not disturb granulocytes activity and stimulate immunological angiogenesis and, accordingly, could be used as a safe drug for therapeutic angiogenesis in vascular and immune system disturbances . Padma 28 tablets (batch 28/6311, padma ag, suisse), herbal mixture consisting of 22 ingredients: aegle marmelos fruit (20 mg), pimenta dioica fruit (25 mg), aquilegia vulgaris aerial part (15 mg), calendula officinalis flower (5 mg), elettaria cardamomum fruit (30 mg), syzygium aromaticum flower bud (12 mg), saussurea lappa root (40 mg), hedychium spicatum rhizome (10 mg), lactuca sativa leaf (6 mg), cetraria islandica thallus (40 mg), glycyrrhiza glabra root (15 mg), azadirachta indica fruit (35 mg), terminalia chebula fruit (30 mg), plantago lanceolata aerial part (15 mg), polygonum aviculare aerial part (15 mg), potentilla aurea aerial part (15 mg), pterocarpus santalinus wood (30 mg), sida cordifolia aerial part (10 mg), aconitum napellus tuber (1 mg), valeriana officinalis root (10 mg), camphor (4 mg), and calcium sulfate (20 mg). The study was performed on 48 female inbred balb / c mice 68 weeks old, weighing about 20 g, and on 24 female f1 hybrids (balb / c c3h), 6 weeks old, delivered from the polish academy of sciences breeding colony . Padma 28 was administered to mice per os in daily doses 5.8 mg or 0,085 mg . Higher dose was calculated according to the highest daily dose (6 tablets), recommended for humans (applying the factor 7 for differences between mouse and human in relation to the surface to body mass). Lower dose conforms to the range of active doses of other herbal extracts and their polyphenolic compounds used in our previous experiments [3135]. Balb / c mice were fed with padma (5.8 mg or 0.085 mg) by eppendorf pipette, in 40 l of water or 40 l of water (controls), for 7 days, then bled in anaesthesia (ketamine 100 mg / kg and xylazine 10 mg / kg, biowet, pulawy, poland) and sacrificed by cervical dislocation . Splenocytes were isolated from spleens of balb / c donors under sterile conditions by straining through stainless sieve and cotton gauze and centrifugation on histopaque 1077 (sigma - aldrich, usa) for 8 min at 400 g in order to remove erythrocytes . Isolated splenocytes were resuspended in parker culture medium (tc199, biomed, lublin) and pooled within the groups . A local gvh reaction (lymphocyte - induced angiogenesis, lia test) shortly, spleen cells suspensions were grafted intradermally (1 million cells in 0.05 ml of parker medium per graft) into f1 (balb / c c3h) recipients . Before performing injections, mice were anaesthetized intraperitoneally with 3.6% chloral hydrate (sigma - aldrich, usa; 0.1 ml per 10 g of body mass). Both flanks of each mouse were finely shaved with a razor blade; each flank was injected with cells 2 - 3 times . Cell suspensions were supplemented with 0.05 ml / ml of 0.01% trypan blue in order to facilitate recognition of injection sites later on . Grafted balb / c splenic lymphocytes recognized c3h antigens and produced many immunological mediators including proangiogenic factors (immunological angiogenesis). In this test, the number of newly formed blood vessels was the measure of t - cell reactivity . After 72 hours the mice were treated with a lethal dose of morbital (biowet, puawy, poland). All newly formed blood vessels were identified and counted in dissection microscope on the inner skin surface, using criteria suggested by the authors of the method, at magnification of 6x, in 1/3 central area of microscopic field . Identification was based on the fact that new blood vessels, directed to the point of cells injection, are thin and differ from the background vasculature in their tortuosity and divarications . Experiment was performed twice (24 balb / c mice and 24 f1 hybrids as a total). Balb / c mice were fed with padma (5.8 mg or 0.085 mg) by eppendorf pipette, in 40 l of water or 40 l of water (controls), for 7 days, then bled in anaesthesia (ketamine 100 mg / kg and xylazine 10 mg / kg, biowet pulawy, poland) from retroorbital plexus and sacrificed by cervical dislocation . Cl was measured using the method of easmon et al . With some modifications [3840] at room temperature, in scintillation counter (rackbeta 1218, lkb, sweden). Briefly, samples of 0.05 ml of heparinised blood were diluted 1: 4 with phosphate buffered saline (pbs, biomed lublin, poland) and supplemented with 0.1% bovine serum albumin (bsa, sigma - aldrich, usa) and 0.1% glucose (polfa, poland). Next, 0.05 ml of this diluted blood was mixed with 0.2 ml of luminol (sigma - aldrich, usa) solution (10 m) in pbs and placed in a scintillation counter in the out of coincidence then, the cells were activated by the addition of 0.02 ml solution of opsonized zymosan (10 mg / ml, serva, usa), and chemiluminescence activity was measured for the next 15 min . Counting of leukocytes and blood smears examination were performed by routine methods, and the results were shown as the maximum value of chemiluminescence (cpm) obtained for 10 granulocytes . For all experiments, animals were handled according to the polish law on the protection of animals and nih standards . All experiments were accepted by the local ethical committee . Statistical evaluation of the results was performed by one - way anova, and the significance of differences between the groups was verified with a bonferroni multiple comparison post test (graph pad prism software package). The effect of padma 28 (0.085 or 5.8 mg) supplementation of balb / c donors on the angiogenic ability of their splenic lymphocytes to induce newly formed blood vessels in the skin of f1 (balb / c c3h) recipient mice is presented in figure 1 . According to one - way analysis of variance the p value <0.0001 bonferroni multiple comparison test revealed that padma 28 in both doses highly significantly stimulated lymphocytes angiogenic activity as compared to the control and that this stimulation was better after higher dose of remedy (for comparison of lower and higher doses, p <0.01). The results of granulocytes chemiluminescence are presented in figure 2 . According to one - way analysis of variance, bonferroni multiple comparison test revealed significantly lower chemiluminescence of blood leukocytes collected from mice fed with higher padma 28 dose, as compared to the control . The results of padma 28 feeding on the blood granulocytes number are presented in figure 3 . According to one - way analysis of variance the p value 0.0006 bonferroni test revealed highly significantly statistical increase of blood granulocytes number in the blood collected from mice fed with lower padma 28 dose in comparison to the control (p <0.001) and significant increase (p <0.05) in comparison to the group fed with high dose of remedy . Bonferroni test revealed highly significant increase of blood lymphocytes number in the blood collected from mice fed with lower padma 28 dose (p <0.001) in comparison to the control and significant increase (p <0.05) in comparison to the group fed with high dose of remedy . In this paper, we report for the first time stimulatory effect of multiherbal remedy padma 28 on immunological angiogenesis observed in the skin of recipient mice during the local cutaneous graft - versus - host reaction . In our previous studies we obtained similar effects, when we administered to donor mice some other herbal extracts (from plants echinacea purpurea, pallida, and angustifolia and rhodiola rosea, quadrifida, and kirilowii) and remedies echinasal and bioaron c. [3135]. However, no stimulatory effect was obtained after feeding donor mice with extract from centella asiatica or multicomponent herbal remedy pervivo [31, 41]. On the other hand, inhibitory effect was observed when donor mice were fed with fibs an aqueous solution of biogenic stimulators (coastal salt lake mud distillate mixed with cinnamic acid and coumarin) elaborated in 1948 year by professor v. p. filatov team . In our present experiments padma 28 has behaved as a strong stimulator of proangiogenic factors production by splenic lymphocytes in mice . It remains to elucidate which factors are involved, and this will be the matter of our further studies . It is important that padma exerted this angiostimulatory effect also in a substantially lower dose than that recommended by producer, because higher recommended dose significantly inhibited granulocyte respiratory burst measured by chemiluminescence . It should be expected as padma 28 contains many compounds demonstrating antioxidant effects gallic acid, eugenol, ellagitannins, bioflavonoids: quercetin, luteolin, and apigenin, and others . The ability of padma 28 to increase angiogenic activity of lymphocytes may partly explain its beneficial effect on regenerative / repair processes, but for this purpose this remedy should be used in evidently lower doses than these recommended by producers, for avoiding deterioration of granulocyte function.
Racially / ethnically diverse individuals with diabetes diagnosed at age <18 years and not due to another condition were recruited through clinics, health fairs, and mailings . One hundred probands with childhood - onset diabetes have participated with their families to date; this analysis was restricted to those with type 1 diabetes (n = 79). Examinations were conducted in the morning in the university of chicago general clinical research center or in participants' homes . Participants aged 18 years and parents of children aged <18 years provided written consent; children 1017 years old assented . Current age, pubertal stage, race / ethnicity, and head of household's education and employment were collected by questionnaire . Pubertal stage was self - assessed with pubic hair tanner diagrams, with missing values (n = 9) imputed using age-, sex-, and race / ethnicity - specific estimates (11). Proband race / ethnicity was defined as that reported for 3 grandparents; if <3 grandparents shared the same race / ethnicity, the proband was of mixed origin . When race / ethnicity was available on <3 grandparents, parental data were used . If parental data were missing, proband's self - reported race / ethnicity was used . Height was measured using a stadiometer; percent body fat (10 years old) and weight were measured with a bioelectrical impedance analyzer scale (tbf-300a; tanita, arlington heights, il). Bmi was transformed into z scores using the centers for disease control and prevention growth chart (20 years old) (12) and national health and nutrition examination survey iii (> 20 years old) (13) age - and sex - matched reference data . Proband overweight / obese was defined as a bmi z score 1.04 (20 years old) or bmi 25 kg / m (> 20 years old) (12,13). Age at diagnosis was abstracted from medical records; if unavailable, it was self - reported as were frequency of insulin injections and parental diabetes . Participants reporting 3 injections per day or use of a pump were defined as receiving an intensive insulin regimen . Type 1 diabetes was defined as having 1) no c - peptide (n = 69) or 2) detectable c - peptide with <2 years duration and either positive islet autoantibodies (gad, insulinoma - associated protein 2; n = 10) or receiving intensive insulin therapy (n = 1). Fasting plasma c - peptide was measured in all probands . Those with a fasting blood glucose <150 mg / dl, measured by a glucometer (onetouch surestep; lifescan, milpitas, ca), also had a stimulated plasma c - peptide measurement 90 min after ingestion of a 6 ml / kg standard nutrient solution (boost; novartis nutrition, minneapolis, mn). C - peptide in probands whose fasting glucose was 150 mg / dl was considered stimulated . C - peptide was determined with a solid - phase, competitive chemiluminescent enzyme immunoassay (immulite 2000; diagnostic products, bad nauheim, germany) in the university of chicago diabetes research and training center laboratory . Nmol / l, and the intra - assay coefficient of variation (cv) was 8% . Absent c - peptide was defined as a fasting, and stimulated if measured, level below the detection limit . Antibodies to radiolabeled recombinant human gad65 (whole) and human insulinoma - associated protein 2 (349 amino acid cytoplasmic portion) were quantified by a fluid - phase immunoprecipitation assay . Dyslipidemia was defined by 1) use of lipid - lowering medications and/or 2) for participants aged 20 years, total cholesterol 200 mg / dl, triglycerides 150 mg / dl, ldl 100 mg / dl, or hdl 59 mg / dl; and for participants aged <20 years, total cholesterol 170 mg / dl, triglycerides 150 mg / dl, ldl 110 mg / dl, or hdl 34 mg / dl . Blood pressure was measured three times; the mean of the second and third values was used . Hypertension was defined by 1) use of antihypertensive medications and/or 2) systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg for participants aged 18 years old or systolic and/or diastolic blood pressure exceeding the age-, sex-, and height - specific 95th percentile (14) for those aged <18 years . Nephropathy was defined by 1) use of ace inhibitors for kidney dysfunction and/or 2) an albumin - to - creatinine ratio 30 mg / g . The michigan neuropathy screening instrument examination was administered by a physician; participants completed the instrument's questionnaire . Neuropathy was defined by 1) a questionnaire score 5 and/or 2) an examination score> 2 (15). Fasting serum insulin was measured with a solid - phase, two - site chemiluminescent immunometric assay (immulite 1000; siemens medical solutions diagnostics, los angeles, ca) by the diabetes research and training center laboratory . The dca 2000 + analyzer (bayer healthcare, elkhart, in) was used to measure a1c in whole blood and urinary albumin and creatinine . A1c was measured using a latex immunoagglutination inhibition method, and the intra- and interassay cvs were 4.3% . Albumin and creatinine were measured through a benedict - behre chemical reaction, and the intra- and interassay cvs were 6.6% . Total cholesterol and hdl were measured in whole blood by the cholestech ldx system using reflectance photometry (cholestech, hayward, ca). Triglycerides were also measured by the cholestech ldx system by an enzymatic method . For all cholesterol measures, c - reactive protein was measured by the university of chicago clinical laboratory using the crplx immunoturbidimetric assay (roche diagnostics, indianapolis, in). The cot one step cotinine test device (quiktest usa, boca raton, fl) was used to detect urine cotinine using a lateral flow chromatographic immunoassay . Serum leptin was determined by the diabetes research and training center laboratory using the human leptin radioimmunoassay kit (millipore, st . Serum adiponectin was measured using the human adipokine panel a-3 plex assay (millipore) by dr . Plasma total and free testosterone and sex hormone binding globulin (shbg) were measured in those aged 10 years old by the university of chicago endocrine laboratory . Total and free testosterone were determined by a solid - phase i radioimmunoassay (siemens medical solutions diagnostics). Insulin resistance in probands was assessed using egdr, a measure derived from clamp studies in 24 nhw adults with long - standing type 1 diabetes using hypertension status, waist - to - hip ratio, and glycemic control (5). Because hip circumference was not available, the comparable equation with waist circumference was provided: egdr (mg kg min) = 21.158 [3.407 hypertension status (yes = 1; no = 0)] [0.090 waist circumference (cm)] [0.551 a1c (%)]. Egdr was highly correlated with clamp - measured insulin resistance (r = 0.79), and egdr calculated using waist - to - hip ratio or waist circumference was highly correlated (r = 0.87) (t.j . The range of clamp - measured glucose disposal rates in the egdr validation study was 3.8 to 13.4, with a range of 9 to 11 in those with normal insulin resistance (5). With the use of fasting insulin and glucose, insulin resistance was determined in the parents (excluding four with type 1 diabetes and two pregnant mothers) by homeostasis model assessment, version 2.0 (17). Analyses were performed in sas (version 9.1; sas institute, cary, nc); statistical tests were considered significant at p <0.05 . Racial / ethnic groups were compared using t tests for continuous and tests for categorical variables . The nonwhite groups (nhb, other / mixed, and hispanic) were combined owing to small sample sizes and similar mean egdr . Triglycerides, albumin - to - creatinine ratio, and leptin were log - transformed . Associations of egdr with hypertension, waist circumference, and a1c were not studied because they were used to calculate egdr; the association with bmi z score was not analyzed because it was highly correlated with waist circumference (r = 0.92, p <0.0001). The multivariable model estimating egdr was built by first entering all variables with p <0.15 from the age - adjusted regressions and then using a stepwise approach to remove the nonsignificant covariates . Age - adjusted logistic regression tested the difference in hypertension prevalence by race / ethnicity . Current age, pubertal stage, race / ethnicity, and head of household's education and employment were collected by questionnaire . Pubertal stage was self - assessed with pubic hair tanner diagrams, with missing values (n = 9) imputed using age-, sex-, and race / ethnicity - specific estimates (11). Proband race / ethnicity was defined as that reported for 3 grandparents; if <3 grandparents shared the same race / ethnicity, the proband was of mixed origin . When race / ethnicity was available on <3 grandparents, parental data were used . If parental data were missing, proband's self - reported race / ethnicity was used . Height was measured using a stadiometer; percent body fat (10 years old) and weight were measured with a bioelectrical impedance analyzer scale (tbf-300a; tanita, arlington heights, il). Bmi was transformed into z scores using the centers for disease control and prevention growth chart (20 years old) (12) and national health and nutrition examination survey iii (> 20 years old) (13) age - and sex - matched reference data . Proband overweight / obese was defined as a bmi z score 1.04 (20 years old) or bmi 25 kg / m (> 20 years old) (12,13). Age at diagnosis was abstracted from medical records; if unavailable, it was self - reported as were frequency of insulin injections and parental diabetes . Participants reporting 3 injections per day or use of a pump were defined as receiving an intensive insulin regimen . Type 1 diabetes was defined as having 1) no c - peptide (n = 69) or 2) detectable c - peptide with <2 years duration and either positive islet autoantibodies (gad, insulinoma - associated protein 2; n = 10) or receiving intensive insulin therapy (n = 1). Fasting plasma c - peptide was measured in all probands . Those with a fasting blood glucose <150 mg / dl, measured by a glucometer (onetouch surestep; lifescan, milpitas, ca), also had a stimulated plasma c - peptide measurement 90 min after ingestion of a 6 ml / kg standard nutrient solution (boost; novartis nutrition, minneapolis, mn). C - peptide in probands whose fasting glucose was 150 mg / dl was considered stimulated . C - peptide was determined with a solid - phase, competitive chemiluminescent enzyme immunoassay (immulite 2000; diagnostic products, bad nauheim, germany) in the university of chicago diabetes research and training center laboratory . Nmol / l, and the intra - assay coefficient of variation (cv) was 8% . Absent c - peptide was defined as a fasting, and stimulated if measured, level below the detection limit . Antibodies to radiolabeled recombinant human gad65 (whole) and human insulinoma - associated protein 2 (349 amino acid cytoplasmic portion) were quantified by a fluid - phase immunoprecipitation assay . Dyslipidemia was defined by 1) use of lipid - lowering medications and/or 2) for participants aged 20 years, total cholesterol 200 mg / dl, triglycerides 150 mg / dl, ldl 100 mg / dl, or hdl 59 mg / dl; and for participants aged <20 years, total cholesterol 170 mg / dl, triglycerides 150 mg / dl, ldl 110 mg / dl, or hdl 34 mg / dl . Blood pressure was measured three times; the mean of the second and third values was used . Hypertension was defined by 1) use of antihypertensive medications and/or 2) systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg for participants aged 18 years old or systolic and/or diastolic blood pressure exceeding the age-, sex-, and height - specific 95th percentile (14) for those aged <18 years . Nephropathy was defined by 1) use of ace inhibitors for kidney dysfunction and/or 2) an albumin - to - creatinine ratio 30 mg / g . The michigan neuropathy screening instrument examination was administered by a physician; participants completed the instrument's questionnaire . Neuropathy was defined by 1) a questionnaire score 5 and/or 2) an examination score> 2 (15). Fasting serum insulin was measured with a solid - phase, two - site chemiluminescent immunometric assay (immulite 1000; siemens medical solutions diagnostics, los angeles, ca) by the diabetes research and training center laboratory . The dca 2000 + analyzer (bayer healthcare, elkhart, in) was used to measure a1c in whole blood and urinary albumin and creatinine . A1c was measured using a latex immunoagglutination inhibition method, and the intra- and interassay cvs were 4.3% . Albumin and creatinine were measured through a benedict - behre chemical reaction, and the intra- and interassay cvs were 6.6% . Total cholesterol and hdl were measured in whole blood by the cholestech ldx system using reflectance photometry (cholestech, hayward, ca). C - reactive protein was measured by the university of chicago clinical laboratory using the crplx immunoturbidimetric assay (roche diagnostics, indianapolis, in). The cot one step cotinine test device (quiktest usa, boca raton, fl) was used to detect urine cotinine using a lateral flow chromatographic immunoassay . Serum leptin was determined by the diabetes research and training center laboratory using the human leptin radioimmunoassay kit (millipore, st . Serum adiponectin was measured using the human adipokine panel a-3 plex assay (millipore) by dr . Plasma total and free testosterone and sex hormone binding globulin (shbg) were measured in those aged 10 years old by the university of chicago endocrine laboratory . Total and free testosterone were determined by a solid - phase i radioimmunoassay (siemens medical solutions diagnostics). Insulin resistance in probands was assessed using egdr, a measure derived from clamp studies in 24 nhw adults with long - standing type 1 diabetes using hypertension status, waist - to - hip ratio, and glycemic control (5). Because hip circumference was not available, the comparable equation with waist circumference was provided: egdr (mg kg min) = 21.158 [3.407 hypertension status (yes = 1; no = 0)] [0.090 waist circumference (cm)] egdr was highly correlated with clamp - measured insulin resistance (r = 0.79), and egdr calculated using waist - to - hip ratio or waist circumference was highly correlated (r = 0.87) (t.j . The range of clamp - measured glucose disposal rates in the egdr validation study was 3.8 to 13.4, with a range of 9 to 11 in those with normal insulin resistance (5). With the use of fasting insulin and glucose, insulin resistance was determined in the parents (excluding four with type 1 diabetes and two pregnant mothers) by homeostasis model assessment, version 2.0 (17). Current age, pubertal stage, race / ethnicity, and head of household's education and employment were collected by questionnaire . Pubertal stage was self - assessed with pubic hair tanner diagrams, with missing values (n = 9) imputed using age-, sex-, and race / ethnicity - specific estimates (11). Proband race / ethnicity was defined as that reported for 3 grandparents; if <3 grandparents shared the same race / ethnicity, the proband was of mixed origin . When race / ethnicity was available on <3 grandparents, parental data were used . If parental data were missing, proband's self - reported race / ethnicity was used . Height was measured using a stadiometer; percent body fat (10 years old) and weight were measured with a bioelectrical impedance analyzer scale (tbf-300a; tanita, arlington heights, il). Bmi was transformed into z scores using the centers for disease control and prevention growth chart (20 years old) (12) and national health and nutrition examination survey iii (> 20 years old) (13) age - and sex - matched reference data . Proband overweight / obese was defined as a bmi z score 1.04 (20 years old) or bmi 25 kg / m (> 20 years old) (12,13). Age at diagnosis was abstracted from medical records; if unavailable, it was self - reported as were frequency of insulin injections and parental diabetes . Participants reporting 3 injections per day or use of a pump were defined as receiving an intensive insulin regimen . Type 1 diabetes was defined as having 1) no c - peptide (n = 69) or 2) detectable c - peptide with <2 years duration and either positive islet autoantibodies (gad, insulinoma - associated protein 2; n = 10) or receiving intensive insulin therapy (n = 1). Those with a fasting blood glucose <150 mg / dl, measured by a glucometer (onetouch surestep; lifescan, milpitas, ca), also had a stimulated plasma c - peptide measurement 90 min after ingestion of a 6 ml / kg standard nutrient solution (boost; novartis nutrition, minneapolis, mn). C - peptide in probands whose fasting glucose was 150 mg / dl was considered stimulated . C - peptide was determined with a solid - phase, competitive chemiluminescent enzyme immunoassay (immulite 2000; diagnostic products, bad nauheim, germany) in the university of chicago diabetes research and training center laboratory . Nmol / l, and the intra - assay coefficient of variation (cv) was 8% . Absent c - peptide was defined as a fasting, and stimulated if measured, level below the detection limit . Antibodies to radiolabeled recombinant human gad65 (whole) and human insulinoma - associated protein 2 (349 amino acid cytoplasmic portion) were quantified by a fluid - phase immunoprecipitation assay . Dyslipidemia was defined by 1) use of lipid - lowering medications and/or 2) for participants aged 20 years, total cholesterol 200 mg / dl, triglycerides 150 mg / dl, ldl 100 mg / dl, or hdl 59 mg / dl; and for participants aged <20 years, total cholesterol 170 mg / dl, triglycerides 150 mg / dl, ldl 110 mg / dl, or hdl 34 mg / dl . Blood pressure was measured three times; the mean of the second and third values was used . Hypertension was defined by 1) use of antihypertensive medications and/or 2) systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg for participants aged 18 years old or systolic and/or diastolic blood pressure exceeding the age-, sex-, and height - specific 95th percentile (14) for those aged <18 years . Nephropathy was defined by 1) use of ace inhibitors for kidney dysfunction and/or 2) an albumin - to - creatinine ratio 30 mg / g . The michigan neuropathy screening instrument examination was administered by a physician; participants completed the instrument's questionnaire . Neuropathy was defined by 1) a questionnaire score 5 and/or 2) an examination score> 2 (15). Fasting serum insulin was measured with a solid - phase, two - site chemiluminescent immunometric assay (immulite 1000; siemens medical solutions diagnostics, los angeles, ca) by the diabetes research and training center laboratory . The dca 2000 + analyzer (bayer healthcare, elkhart, in) was used to measure a1c in whole blood and urinary albumin and creatinine . A1c was measured using a latex immunoagglutination inhibition method, and the intra- and interassay cvs were 4.3% . Albumin and creatinine were measured through a benedict - behre chemical reaction, and the intra- and interassay cvs were 6.6% . Total cholesterol and hdl were measured in whole blood by the cholestech ldx system using reflectance photometry (cholestech, hayward, ca). Triglycerides were also measured by the cholestech ldx system by an enzymatic method . For all cholesterol measures, c - reactive protein was measured by the university of chicago clinical laboratory using the crplx immunoturbidimetric assay (roche diagnostics, indianapolis, in). The cot one step cotinine test device (quiktest usa, boca raton, fl) was used to detect urine cotinine using a lateral flow chromatographic immunoassay . Serum leptin was determined by the diabetes research and training center laboratory using the human leptin radioimmunoassay kit (millipore, st . Serum adiponectin was measured using the human adipokine panel a-3 plex assay (millipore) by dr . Plasma total and free testosterone and sex hormone binding globulin (shbg) were measured in those aged 10 years old by the university of chicago endocrine laboratory . Total and free testosterone were determined by a solid - phase i radioimmunoassay (siemens medical solutions diagnostics). Insulin resistance in probands was assessed using egdr, a measure derived from clamp studies in 24 nhw adults with long - standing type 1 diabetes using hypertension status, waist - to - hip ratio, and glycemic control (5). Because hip circumference was not available, the comparable equation with waist circumference was provided: egdr (mg kg min) = 21.158 [3.407 hypertension status (yes = 1; no = 0)] [0.090 waist circumference (cm)] [0.551 a1c (%)]. Egdr was highly correlated with clamp - measured insulin resistance (r = 0.79), and egdr calculated using waist - to - hip ratio or waist circumference was highly correlated (r = 0.87) (t.j . The range of clamp - measured glucose disposal rates in the egdr validation study was 3.8 to 13.4, with a range of 9 to 11 in those with normal insulin resistance (5). With the use of fasting insulin and glucose, insulin resistance was determined in the parents (excluding four with type 1 diabetes and two pregnant mothers) by homeostasis model assessment, version 2.0 (17). Analyses were performed in sas (version 9.1; sas institute, cary, nc); statistical tests were considered significant at p <0.05 . Racial / ethnic groups were compared using t tests for continuous and tests for categorical variables . The nonwhite groups (nhb, other / mixed, and hispanic) were combined owing to small sample sizes and similar mean egdr . Triglycerides, albumin - to - creatinine ratio, and leptin were log - transformed . Associations of egdr with hypertension, waist circumference, and a1c were not studied because they were used to calculate egdr; the association with bmi z score was not analyzed because it was highly correlated with waist circumference (r = 0.92, p <0.0001). The multivariable model estimating egdr was built by first entering all variables with p <0.15 from the age - adjusted regressions and then using a stepwise approach to remove the nonsignificant covariates . Age - adjusted logistic regression tested the difference in hypertension prevalence by race / ethnicity . For the 79 probands (table 1), mean current age was 13.5 years (range 3.232.5); 32.9% were nhw, 46.8% were nhb, 7.6% were other / mixed, and 12.7% were hispanic . Mean disease duration was 5.7 years (0.119.9), mean current a1c was 9.1%, and 69.6% were receiving an intensive insulin regimen . Only six probands were overweight / obese (one nhw, four nhb, and one other / mixed). A large proportion had complications including dyslipidemia (53.2%) and hypertension (21.5%). More than two - thirds had at least one obese parent, and 14.5% had at least one parent who self - reported diabetes . Current age had an inverse association with egdr: egdr (mg kg min) = 12.51 [0.26 age (years)] (p <0.0001). Diabetes duration also had an inverse association with egdr: egdr (mg kg min) = 10.15 [0.19 duration (years)] (p = 0.004). However, the relationship with diabetes duration was not significant after adjusting for age . Race / ethnicity was significantly associated with egdr even after adjustment for age; compared with nhw, egdr was significantly lower among nonwhites [mean difference () = 1.83, p = 0.0006]. Associations of egdr with demographic, anthropometric, diabetes, clinical, and hormonal characteristics in probands with type 1 diabetes n = 79 . With adjustment for age, egdr had a significant negative association with percent body fat (table 2). Probands with at least one obese parent had significantly lower egdr levels than those with no obese parent (= 2.42, p = 0.0001); further adjustment for probands' percent body fat did not attenuate this association . Probands with at least one insulin - resistant parent also had significantly lower egdr than those with no insulin - resistant parent (= 1.79, p = 0.03); however, parental self - reported diabetes status was not associated with proband egdr . Egdr was positively associated with hdl and negatively associated with triglycerides and albumin - to - creatinine ratio . Participants with acanthosis nigricans and those with elevated c - reactive protein had significantly lower egdr compared with those without (= 2.06, p = 0.0002, and = 1.84, p = 0.04, respectively). In those with body fat determined (10 years old, n = 57), the association of egdr with c - reactive protein was also significant, but the relationship became nonsignificant after adjustment for body fat . Both leptin and adiponectin were negatively associated with egdr, but after controlling for age, adiponectin was no longer significant (table 2). Further, for the subgroup of probands with body fat determined (n = 51), leptin was negatively associated with egdr, but subsequent adjustment for percent body fat attenuated this association . In stratified analyses, shbg was positively associated with egdr in both sexes and remained significant in women after controlling for age . Among all probands, there was a significant age- and sex - adjusted positive association between egdr and shbg (regression coefficient = 0.96 mg kg min rise in egdr per 10 nmol / l increase in shbg, p = 0.0001); additional adjustment for proband percent body fat did not change this association . In multivariable analysis (table 3), lower egdr was significantly associated with older age, nonwhite race / ethnicity, and acanthosis . When hdl was included, resulting in a smaller sample size, these associations remained significant . The potential confounding effects of probands' percent body fat, parental obesity, and parental insulin resistance on these associations were examined and found not to occur nor did race / ethnicity modify the associations of egdr with these factors . Variables entered into the model: current age, race / ethnicity, head of household education, percent body fat, parental obesity, parental insulin resistance, hdl, triglycerides, albumin - to - creatinine ratio, acanthosis, elevated c - reactive protein, positive cotinine, sleep duration, and shbg . Whether the racial / ethnic variation in egdr was driven by differences in the components used to calculate egdr was explored . With adjustment for age, compared with nhw participants, nonwhites had significantly higher a1c and a trend for larger waist circumference (9.7 vs. 7.9%, p = 0.0005, and 72.2 vs. 68.2 cm, p = 0.10, respectively; estimates at mean age of 13.5 years), and a trend toward a higher prevalence of hypertension (26.4 vs. 11.5%, odds ratio 3.1, p = 0.10). Insulin resistance is an important component of type 1 diabetes (2). To our knowledge, this is the first study to show that racial / ethnic differences in insulin resistance, as measured by egdr, exist in this group . Furthermore, this variation was observed in a sample of young individuals with short diabetes duration and normal adiposity, suggesting that racial / ethnic differences are present early in the disease course . The current results are consistent with previous research in healthy individuals and in those with type 2 diabetes of whom nonwhite groups were generally more insulin resistant . Specifically, nondiabetic african american and hispanic adults were more insulin resistant than nondiabetic nhw adults, using a frequently sampled intravenous glucose tolerance test with minimal model analysis (8). For adults with type 2 diabetes, african americans, but not hispanics, were more insulin resistant than nhw (1). The picture may be more complicated in adolescence, because puberty is an insulin - resistant state (10). One study demonstrated that mexican american, but not black, children were more insulin - resistant than nhw youth as measured by homeostasis model assessment (9), whereas another study using the clamp method found that insulin resistance was increased in black but not white participants between 11 and 19 years of age (10). Our results demonstrating trends for racial / ethnic differences in the egdr components are also consistent with the literature, with minorities demonstrating poorer outcomes . For example, african american children with type 1 diabetes have worse glycemic control than white children with diabetes (18). With regard to adiposity, a higher percentage of mexican - american and black adolescents are overweight / obese compared with nhw adolescents (9). The national health and nutrition examination survey iii also found that african american adult women with type 2 diabetes have a higher bmi than nhw women with diabetes (19). Last, hypertension is more prevalent in african american adults than in nhw and mexican american adults, both with and without type 2 diabetes (20). For example, higher insulin resistance is related to older age and longer disease duration (6). The authors found similar relationships, but the association with age was stronger and duration was no longer significant after adjustment for age . Family history of type 2 diabetes has been shown to be associated with greater insulin resistance in those with type 1 diabetes (5). In the current study, there was no association with parental history of diabetes, which was based on self - report . However, parental obesity and parental insulin resistance, which were directly measured in the current study and are risk factors for diabetes, were related to greater proband age - adjusted insulin resistance . This association was independent of the probands' adiposity, indicating that other familial characteristics in addition to factors affecting probands' body composition (e.g., genetic predisposition) may influence their insulin resistance . Lower levels of shbg were also significantly associated with greater insulin resistance in age - adjusted analysis . This finding is consistent with basic science (21) and clinical research (22) demonstrating a negative association between insulin and shbg levels, such that the greater the level of insulin resistance in an individual with type 1 diabetes is, the higher is the insulin dose required to meet physiological requirements and the lower the hepatic production of shbg . These results suggest that the derived measure of insulin resistance, egdr, is indeed measuring insulin resistance in those with type 1 diabetes . The risk of micro - and macrovascular complications is elevated for individuals with type 1 diabetes who have insulin resistance . For example, greater insulin resistance is associated with the development of nephropathy (3,6), neuropathy (7), and elevated lipids (6). In the current study, it could be argued that these relationships may be capturing the well - established associations of diabetes complications with the components used to calculate egdr, specifically suboptimal glycemic control and/or hypertension . However, previous research in nhw adults with long - standing type 1 diabetes showed that insulin resistance as measured by egdr more strongly predicted overt diabetic nephropathy than did glycemia or blood pressure (23). Egdr is a derived measure of insulin resistance, and clamp techniques are needed to confirm our results . However, because egdr is noninvasive and strongly correlated with clamp - measured insulin resistance, it is useful for population - based studies, similar to the numerous surrogate measures of insulin resistance that are commonly used in studies of type 2 diabetic and nondiabetic groups . We demonstrated for the first time associations of proband insulin resistance with parental obesity and parental insulin resistance, although there may have been limited power to confirm the associations in the multivariable model . Limited sample size may also explain the lack of a significant difference in insulin resistance by neuropathy status, although the magnitude of the difference was small . Last, because we combined nonwhite groups owing to small sample sizes and similar egdr, we may have limited the ability to detect interactions by race / ethnicity . The research had a number of strengths, most importantly that it is the first study of type 1 diabetes, to our knowledge, to examine insulin resistance using a racially / ethnically diverse sample . We were also able to evaluate and adjust for confounders in the multivariable model to estimate the independent association of race / ethnicity with insulin resistance . The greater insulin resistance observed in nonwhites with type 1 diabetes may help clarify the well - documented gaps in diabetes outcomes for underserved minorities . Insulin resistance is associated with a spectrum of poor health outcomes; thus, the greater insulin resistance in minorities with type 1 diabetes may explain their higher incidence of diabetes - related complications compared with nhw (24). Future research is needed to determine which factors contribute to racial / ethnic differences in insulin resistance in this group, such as disparities in diabetes care, differences in behaviors, environmental factors, or physiological processes, or simply residual confounding . Understanding which modifiable factors prevent insulin resistance and then targeting interventions to those groups most at risk will help address the public health burden of type 1 diabetes and its complications in minority populations . In summary, insulin resistance as estimated by egdr was greater in minorities with type 1 diabetes than in nhw probands . Because there exists a strong association between insulin resistance and poor health outcomes in diabetes, the search for effective interventions to improve insulin resistance in minorities with type 1 diabetes must become a priority.
In a laboratory experiment previously reported (9), the first generation (f1) larval progeny of engorged a. aureolatum female ticks collected in atibaia, so paulo state, brazil, were allowed to feed on 4 r. rickettsii infected guinea pigs (infected group) and 2 uninfected guinea pigs (control group). These guinea pigs were infected by intraperitoneal inoculation of a homogenate of r. rickettsii infected guinea pig organs, as detailed previously (9). The taiau strain of r. rickettsii used in this experiment was the first guinea pig passage from a naturally infected a. aureolatum tick, which was cryopreserved before this strain was adapted for in vitro growth (5). Engorged tick larvae obtained from the infected and control groups of guinea pigs were held in an incubator at 23c and 90% relative humidity for molting to nymphs . As reported (9), 100% of the resultant nymphs from the infected group were shown to be infected by r. rickettsii, whereas no nymph from the control group was infected by rickettsiae . The f1 unfed nymphs of infected and control groups were reared separately in the laboratory for 4 consecutive generations until they were f4 unfed adults . Throughout the experiment, infestations with infected ticks and control groups were done in parallel; infested animals were held in the same room under the same environmental conditions as the controls . Male guinea pigs and female rabbits were infested with larvae and nymphs, and dogs and rabbits were infested with adult ticks . However, in each infestation with a given tick stage, different individuals of the same host species were used at the same time for infected and control groups . Every guinea pig or rabbit used in this study was tick naive; these animals were provided by a private laboratory that raised the animals under proper sanitary conditions . The dogs used for adult infestations had been infested by a. aureolatum ticks in a previous study (10), when they were also infected with r. rickettsii; the dogs were therefore immune to rmsf . Infestation of each animal was performed inside a feeding chamber glued to its shaved dorsum, as previously described (11). All infested animals had their temperature rectally measured daily from the day of infestation (day 0) to 21 days postinfestation . When present, skin lesions (e.g., scrotal reactions) during this period were also noted . Naturally detached engorged larvae, nymphs, or female ticks were recovered daily from the feeding chambers of the infested animals of both groups and immediately taken to a single incubator adjusted to 23c and 95% relative humidity for molting (for engorged larvae and nymphs) or for egg laying and incubation (for engorged females). Engorged females had their individual weight measured the day they detached from the host . In addition, the total egg mass deposited by each female was weighed on the day of the end of oviposition and a conversion efficiency index (cei = mg egg mass / mg engorged female 100), which measures the efficiency with which a female tick converts body weight into eggs (12), was determined for each female that oviposited . Percentage of egg hatching for each egg mass was visually estimated (13). During the experiment, random samples of unfed f1 nymphs and adults; f2 eggs and nymphs; and f4 eggs, larvae, nymphs, and adults from both infected and control groups were submitted for dna extraction as described (9) and tested by pcr targeting a 401-bp fragment of the rickettsial glta gene, as described (9). Eggs were tested in pools (1 egg pool containing 510 eggs / female), whereas larvae, nymphs, and adults were tested individually . At the end of the study, pcr products from 3 f4 nymphs underwent dna sequencing (5), and the resultant sequence was compared with genbank data by blast analysis (www.ncbi.nlm.nih.gov/blast). All tick - infested guinea pigs and rabbits were tested for seroconversion to r. rickettsii antigens . For this purpose, blood samples were collected at 0 days postinfestation and 21 days postinfestation; these samples were tested for anti rickettsii reactive antibodies by immunofluorescence assay (ifa), as previously described (14). Some infested guinea pigs that died before the 21 days postinfestation were not tested by ifa because a second blood sample was not obtained; however, a fragment of their lungs was submitted to dna extraction by using the dneasy tissue kit (qiagen, chatsworth, ca, usa) and tested by the same pcr protocol referenced above . During the experiment, tick biologic parameters were compared between infected and control groups . For this purpose, larval and nymphal molting success and female oviposition success (i.e., death of engorged ticks) were compared by the test . In addition, weight of engorged females and their corresponding egg masses, percentage of egg hatching, and cei values were compared by student t test . The study was approved by the bioethical committee in animal research of the faculty of veterinary medicine of the university of so paulo (protocol no . The infected tick group remained infected by rickettsiae through 4 consecutive generations, until the end of the experiment (f4 unfed adults). In all infestations performed with ticks from this group, fever developed in all guinea pigs and rabbits 59 days postinfestation . Guinea pigs also had scrotal reactions characterized by swelling, followed by necrosis in most animals . Among the 16 guinea pigs infested with infected larvae or nymphs, 8 died during the febrile period . Of the 8 dead guinea pigs, 6 had their lungs tested by pcr, which detected rickettsial dna . The remaining guinea pigs plus the rabbits showed seroconversion for r. rickettsii at 21 days postinfestation (table 1). In contrast, no guinea pig or rabbit infested with ticks from the control group experienced fever or seroconverted; i.e., they were nonreactive for r. rickettsii at both 0 days postinfestation and 21 days postinfestation . Fever did not develop in the dogs infested with generations f1 and f3 adult ticks, and serum was not tested on 0 days postinfestation and 21 days postinfestation because it was already known that the dogs were seroreactive to r. rickettsii because they had been previously infected in another study (10). * dpi, days postinfestation; ifa, immunofluorescence assay; pos, positive; nd, not done;, data not collected . Pcr performed on lung - extracted dna from guinea pigs that died during the febrile period . A positive result means seroconversion, namely nonreactive (titer <64) at 0 dpi, and reactive (titer> 1,024) at 21 dpi . All pcrs performed on unfed ticks from the infected group resulted in amplicons compatible with r. rickettsii . These amplicons resulted from 45 f1 nymphs, 10 f1 adults (5 males, 5 females), 30 f2 nymphs, 100 f4 larvae, 30 f4 nymphs, and 6 f4 adults (3 males, 3 females), which were all tested individually (table 1). In addition, 12 f2 egg pools and 5 f4 egg pools, derived from all f1 and f3 infected engorged females, also yielded pcr amplicons compatible with r. rickettsii . The 100 pcr - positive f4 larvae cited above encompassed 5 groups of 20 larvae, each derived from a different f3 engorged female . Pcr products from 3 f4 nymphs underwent dna sequencing, and the resultant sequence was 100% identical to r. rickettsii strain taiau, available in genbank (accession no . No visible amplicon was generated by all ticks tested by pcr, which included 20 f1 nymphs, 6 f1 adults (3 males, 3 females), 10 f2 nymphs, 30 f4 larvae, 20 f4 nymphs, and 6 f4 adults (3 males, 3 females), which were all tested individually, and 13 f2 egg pools and 6 f4 egg pools, derived from all f1 and f3 control engorged females . The 30 pcr - negative f4 larvae cited above encompassed 3 groups of 10 larvae, with each group derived from a different f3 engorged female . Although the mortality rate for engorged f4 larvae and f3 nymphs was higher for the infected group than the control group, overall molting success of engorged larvae and nymphs was similar between infected and control ticks because there was no significant difference in the molting success of these ticks when the 4 generations were grouped . However, there was an overall lower egg - laying success of engorged females from the infected group when compared with the control group (table 2); i.e., although 89.7% of the control engorged females successfully oviposited in the incubator, only 66.7% of the infected females oviposited in the same incubator . Regarding the reproductive performance of these females, a few significant differences were observed between infected and control females, generally in favor of control ticks (table a1). * molting or oviposition success values for infected and control ticks of the same tick stage were significantly different (p<0.05). In the study reported here, r. rickettsii was preserved by transstadial maintenance and transovarial transmission in a. aureolatum ticks for 4 consecutive generations, because all tested eggs, larvae, nymphs, and adults from the infected group were shown by pcr to contain rickettsial dna, from the first to the fourth generation . In addition, infestations of guinea pigs and rabbits with larvae and nymphs from the 4 generations and adults from the third generation confirmed that ticks of these 3 developmental stages from the 4 generations were infected by r. rickettsii because all infested guinea pigs and rabbits became infected by r. rickettsii, which was confirmed by seroconversion through ifa or pcr in addition to compatible clinical data . These results also confirm that larvae, nymphs, and adults of a. aureolatum ticks are competent vectors of r. rickettsii . Together, our results strongly support clinical and epidemiologic data that have implicated the a. aureolatum tick as the main vector of r. rickettsii in the metropolitan area of so paulo (4,5,9,15). All infected egg pools tested by pcr yielded rickettsial dna, indicating a transovarial transmission rate (the proportion of infected females giving rise to at least 1 infected egg or larva) of 100% among r. rickettsii infected females . Because all the individual larvae from the infected group tested by pcr also yielded rickettsial dna, a filial infection rate (proportion of infected eggs or larvae obtained from an infected female) of 100% is also likely to have occurred . In contrast, no egg pool or individual larva from the control group yielded rickettsial dna by pcr . These results are in agreement with the larval infestations, which resulted in confirmed rickettsiosis in all guinea pigs or rabbits infested by larvae from the infected group and with no rickettsiosis in animals infested with control group larvae . Although transovarial transmission of spotted fever rickettsiae in ticks seems to occur worldwide (16), including r. rickettsii in new world ticks (1723), few studies have quantified this perpetuation route . Previous studies in the united states demonstrated a 100% transovarial transmission rate and a 100% filial infection rate of r. rickettsii in d. variabilis naturally infected female ticks and in d. andersoni female ticks, either naturally infected or experimentally infected during the larval stage (20,24). In the study reported here, although f2 female ticks fed on susceptible rabbits, f1 and f3 females fed on dogs previously infected by r. rickettsii . As expected, these dogs showed no clinical signs during the study, a condition certainly related to their immune status because they were shown to have high antibody titers against r. rickettsii (data not shown). Regardless of feeding on immune (dogs) or susceptible (rabbits) hosts, 100% transovarial transmission rates and filial infection rates were found for all generations evaluated . Similarly, burgdorfer and brinton (24) observed 100% transovarial transmission rates and filial infection rates for d. andersoni female ticks that fed on either immune or susceptible hosts, resulting in no alteration of biological characteristics of the bacterium . Thus, the host immune status to r. rickettsii does not seem to alter the transovarial transmission of r. rickettsii in ticks . Overall, no expressive differences in mortality rates were observed between engorged immature ticks from the infected and control groups . These results contrast with those of a previous study (21) that reported much higher mortality rates for infected d. andersoni engorged larvae and nymphs (34.9%97.7%) than those observed for uninfected sibling ticks (1.4%2.1%) and also with those of experimental studies on r. conorii in r. sanguineus ticks, in which significant mortality rates of immature ticks were observed when compared with uninfected ticks (25,26). On the other hand, our results demonstrated that the mortality of r. rickettsii infected engorged females was 3 higher than the mortality of uninfected females; i.e., oviposition success was only 66.7% in the former and 89.7% in the latter . In addition, the reproductive performance of uninfected females was significantly higher than that of infected females, as demonstrated by higher egg mass weight for control ticks . Because both the infected and control ticks were siblings derived from the same field - engorged females used to start a laboratory colony, reared under the same laboratory conditions during the whole study, we conclude that the lower survival and reproductive performance of infected females was a result of a deleterious effect caused by the r. rickettsii infection . These results are in agreement with those of a previous study (24), which reported higher mortality of r. rickettsii infected d. andersoni and d. variabilis engorged female ticks and lower egg masses oviposited by these females than by uninfected females . This higher mortality and lower egg mass was attributed to massive rickettsial development in the female body, including the ovaries (24). Tick mortality is much more influential on the tick population when it occurs in engorged females; i.e., although each dead egg, larva, or nymph is only less 1 subsequent larva, nymph, or adult, respectively, in the tick population, a dead engorged female represents thousands of eggs fewer in the following generation . Different field studies in brazil and in the united states have reported that <1% of the dermacentor spp . And a. aureolatum ticks, respectively, are found naturally infected by r. rickettsii within rmsf - endemic areas (1,5,27). On the other hand, the present study demonstrated that besides being highly susceptible to r. rickettsii infection, a. aureolatum ticks are highly efficient in maintaining the infection through 100% transstadial transmission, transovarial transmission, and filial infection rates . The main reason for these contrasting findings is the deleterious effect the r. rickettsii infection causes in ticks, as previously demonstrated for dermacentor spp . Ticks (21,24,27). Therefore, despite of the high susceptibility of a. aureolatum ticks to r. rickettsii infection, the higher mortality and reduced reproductive performance of infected engorged females may contribute to low infection rates among a. aureolatum tick field populations in rmsf - endemic areas of the so paulo metropolitan area, such as the 0.9% infection rate previously reported (5). On the basis of our results, it seems unlikely that a. aureolatum could sustain r. rickettsii infection over multiple successive generations solely by vertical transmission because the number of infected ticks would gradually decrease after each generation . Thus, horizontal transmission through the participation of amplifier vertebrate hosts in the formation of new lineages of infected ticks seems to be crucial for maintenance of r. rickettsii in the rmsf - endemic areas where the a. aureolatum tick is implicated as the principal vector, just as has been reported for rmsf - endemic areas in the united states, where dermacentor spp . Ticks are the vector (21,27). Although natural amplifier hosts are known for dermacentor spp . Ticks in rmsf - endemic areas in the united states (27), and for a. cajennense ticks in areas endemic for brazilian spotted fever in brazil (1), they are not known for a. aureolatum ticks . Further studies should test the natural hosts of a. aureolatum immature tick stages, which are some passerine birds and small rodents (6), for their competence to act as amplifier hosts of r. rickettsii to a. aureolatum ticks . Regarding dogs, the main host for the adult stage of a. aureolatum ticks within the so paulo metropolitan area, even though it has been shown that dogs are capable of having rickettsial infections sufficient to infect other tick species (i.e., they are a competent amplifier host) (23), only adult - stage a. aureolatum ticks feed on dogs . Because transovarial transmission rates are likely to be low or absent when the primary infection of ticks occurs during adult feeding (18,23,24), the epidemiologic influence of dogs as amplifier hosts of r. rickettsii for a. aureolatum ticks is questionable . Therefore, future studies should target potential hosts of the immature stages of a. aureolatum ticks that could act as amplifier hosts.
The introduction of highly active antiretroviral therapy (haart) and antibiotic prophylaxis against pulmonary opportunistic infections have resulted in considerable reduction of morbidity and mortality of this kind of patients . Some of hiv - infected patients have pulmonary opportunistic infections in the haart era associated with different causes including patients who are unaware of their hiv serostatus and do not initiate haart, late stage of hiv infection, cd4 count less than 100 cells/l, haart non - adherence or failure to respond, injection drug use and failure to provide prophylaxis against opportunistic pathogens . Pulmonary toxoplasmosis is the second location of extracerebral toxoplasmosis after the ocular form, with 40% of mortality without early diagnosis and specific treatment . Pulmonary toxoplasmosis accounts for 4% of all cases of pneumonia in hiv - infected patients in the pre - haart era and is not considered commonly during the haart era . We described two cases of toxoplasma gondii pneumonia in hiv patients in the haart era . A 41-year - old hiv - positive man was admitted to our hospital because of fever and cough since 5 days . His medical history revealed intravenous drugs addiction, chronic renal failure and hepatitis c infection . He was started on haart based on abacavir, lamivudine and efavirenz during the last year . On examination his temperature was 37c, pulse rate of 100 beats per minute and 20 breaths per minute . Relevant laboratory findings showed an hemoglobin level of 7 g / dl, total white cell count of 3,700 cells / mm and a normal platelet count . Renal dysfunction was evidenced by serum urea of 47 mg / dl and creatinine of 1.8 mg / dl . The cd4 t - cell count was 35 cells/l and the plasma viral load was 203,906 copies / ml . Serologic testing revealed the presence of t. gondii igg antibodies with a positive titer of 1:1024 and igm negative by indirect immunofluorescence . Chest radiograph showing bilateral interstitial infiltrates fever and cough continued 7 days after admission to the hospital . On the eighth day a bronchoalveolar lavage (bal) cytological and microbiological examinations of the bal were negative for bacteria, fungi, pneumocystis jirovecii and mycobacterium tuberculosis . Giemsa - stained smears of the bal were negative for tachyzoite forms of t. gondii . An aliquote of the bal fluid was assayed for t. gondii using the pcr technique for amplification of a fragment corresponding to the b1 gene . Computed tomography (ct) scan of the brain showed marked cerebral atrophy without evidence of focal lesions . He showed a marked improvement in clinical, radiological, and laboratory findings after the seventh day of therapy for t. gondii . He was discharged from the hospital after 15 days of admission in a good clinical condition . He was intravenous drug user and had been diagnosed as hiv seropositive 10 years before . He was discharged from the hospital with improvement in clinical and radiological finding after treatment . The patient was not adhering to treatment with haart and secondary prophylaxis for t. gondii infection . On admission, he was febrile (38c), pulse rate of 100 beats per minute and 18 breaths per minute . Laboratory results showed a white blood cell count of 8,600 cell / mm, an hemoglobin level of 12 g / dl and a normal platelet count . Ct scan of the chest showed pleural effusion in the right lung [figure 2b]. Serologic testing revealed the presence of t. gondii igg antibodies with a positive titer of 1:1024 and igm negative by indirect immunofluorescence . Empiric treatment was initiated with ampicilin plus sulbactam . On the fifth day fever and cough continued . Cytological and microbiological examinations were negative for bacteria, fungi, p. jirovecii and m. tuberculosis . Giemsa - stained smears of the bal were negative for tachyzoite forms of t. gondii . A bal fluid sample was assayed for t. gondii using the pcr technique for amplification of a fragment corresponding to the b1 gene . Ct studies of the brain showed a left temporoparietal periventricular lesion with mass effect on the middle - line structures [figure 3]. (a) chest radiograph showing pleural effusion in right pulmonary lobe (b) computerized tomography showing pleural effusion in right pulmonary lobe computerized tomographic studies of the brain showing evidence of a focal lesion treatment for t. gondii infection was initiated with sulfadiazine plus pyrimethamine and folinic acid . In the following days he was discharged from the hospital after 30 days in a good clinical and neurological condition . A 41-year - old hiv - positive man was admitted to our hospital because of fever and cough since 5 days . His medical history revealed intravenous drugs addiction, chronic renal failure and hepatitis c infection . He was started on haart based on abacavir, lamivudine and efavirenz during the last year . On examination his temperature was 37c, pulse rate of 100 beats per minute and 20 breaths per minute . Relevant laboratory findings showed an hemoglobin level of 7 g / dl, total white cell count of 3,700 cells / mm and a normal platelet count . Renal dysfunction was evidenced by serum urea of 47 mg / dl and creatinine of 1.8 mg / dl . The cd4 t - cell count was 35 cells/l and the plasma viral load was 203,906 copies / ml . Serologic testing revealed the presence of t. gondii igg antibodies with a positive titer of 1:1024 and igm negative by indirect immunofluorescence . Chest radiograph showing bilateral interstitial infiltrates fever and cough continued 7 days after admission to the hospital . On the eighth day a bronchoalveolar lavage (bal) cytological and microbiological examinations of the bal were negative for bacteria, fungi, pneumocystis jirovecii and mycobacterium tuberculosis . Giemsa - stained smears of the bal were negative for tachyzoite forms of t. gondii . An aliquote of the bal fluid was assayed for t. gondii using the pcr technique for amplification of a fragment corresponding to the b1 gene . Computed tomography (ct) scan of the brain showed marked cerebral atrophy without evidence of focal lesions . He showed a marked improvement in clinical, radiological, and laboratory findings after the seventh day of therapy for t. gondii . He was discharged from the hospital after 15 days of admission in a good clinical condition . He was intravenous drug user and had been diagnosed as hiv seropositive 10 years before . He was discharged from the hospital with improvement in clinical and radiological finding after treatment . The patient was not adhering to treatment with haart and secondary prophylaxis for t. gondii infection . On admission, he was febrile (38c), pulse rate of 100 beats per minute and 18 breaths per minute . Laboratory results showed a white blood cell count of 8,600 cell / mm, an hemoglobin level of 12 g / dl and a normal platelet count . Ct scan of the chest showed pleural effusion in the right lung [figure 2b]. Serologic testing revealed the presence of t. gondii igg antibodies with a positive titer of 1:1024 and igm negative by indirect immunofluorescence . Empiric treatment was initiated with ampicilin plus sulbactam . On the fifth day fever and cough continued . Cytological and microbiological examinations were negative for bacteria, fungi, p. jirovecii and m. tuberculosis . Giemsa - stained smears of the bal were negative for tachyzoite forms of t. gondii . A bal fluid sample was assayed for t. gondii using the pcr technique for amplification of a fragment corresponding to the b1 gene . Ct studies of the brain showed a left temporoparietal periventricular lesion with mass effect on the middle - line structures [figure 3]. (a) chest radiograph showing pleural effusion in right pulmonary lobe (b) computerized tomography showing pleural effusion in right pulmonary lobe computerized tomographic studies of the brain showing evidence of a focal lesion treatment for t. gondii infection was initiated with sulfadiazine plus pyrimethamine and folinic acid . In the following days he was discharged from the hospital after 30 days in a good clinical and neurological condition . Spectrum of hiv - related pulmonary complications that can be seen in the haart era include p. jirovecii pneumonia, bacterial pneumonia and interstitial pneumonitis with unknown etiology but not pulmonary toxoplasmosis . Clinical manifestations include febrile illness associated with unspecific respiratory symptoms such as cough and dyspnea . Here, we described two hiv - infected patients with poor adherence to haart and antibiotic prophylaxis, with fever and cough but not with dyspnea or adult respiratory distress syndrome (ards). Kovari and colleagues published two cases in 2010 with unknown hiv status and pulmonary toxoplasmosis in switzerland, one patients with ards and the second with productive cough . Other findings include nodular solitary lesions and pneumothorax . In the first case that we described, chest radiograph showed bilateral interstitial infiltrates and, in the second, the swiss cases chest radiographs showed bilateral diffuse interstitial infiltrates, one of them associated with p. jirovecii . Pulmonary toxoplasmosis occurred in patients with advanced immunodeficiency with cd4 t - cell count less than 100 cells/l . Serological data for the patients showed a rise in igg levels but were negative for igm during the acute episode of pulmonary toxoplasmosis . Diagnosis of pulmonary toxoplasmosis is based on the identification of tachyzoites or dna of t. gondii in samples of bal, biopsy specimens or necropsy . Bal samples should be processed by several methods such as giemsa staining, tissue culture, intraperitoneal inoculation of mice, immunoperoxidase assay and detection of parasite dna by molecular techniques . Giemsa - stained smears of bal fluids from the two patients were negative for tachyzoite forms of t. gondii . Visualization of tachyzoites in bal fluid by giemsa is difficult because of their small size or their small number . T. gondii dna is detected in bal specimens with the use of primers targeting the b1 gene . The b1 gene is highly specific for t. gondii and can detect as few as 10 organisms . Diagnosis of pulmonary toxoplasmosis was established in the two cases with the use of primers targeting the b1 gene in bal and blood samples . Antiparasitic therapy based on sulfadiazine and pyrimethamine was associated with a marked improvement in clinical, radiological, and laboratory findings in our cases . In conclusion, pulmonary toxoplasmosis should be included in the differential diagnosis of hiv positive patients with late stage of hiv infection, cd4 t cell counts less than 100 cells/l, a poor adherence or failure to haart and with fever, cough and pulmonary radiological infiltrates.
We describe the first valve - in - valve corevalve transcatheter aortic valve replacement in the st . Jude toronto stentless porcine aortic valve in the united states, which enabled this 59-year - old patient with a history of bacterial endocarditis and aortic regurgitation to avoid heart transplant with complete resolution of his severe left ventricular dysfunction . While transcatheter aortic valve replacement (tavr) volume has grown substantially worldwide in recent years, our collective experience with transcatheter valve - in - valve placement remains relatively low . The first percutaneous valve - in - valve procedure using the corevalve revalving system for aortic regurgitation in a sorin mitroflow aortic bioprosthesis was reported in 2007 in germany . Since then, other case reports of valve - in - valve procedures to treat a failing aortic valve bioprosthesis have been described, but very few in stentless bioprostheses . The global valve - in - valve registry of 202 patients (mean age 78 years, 53% men) includes 124 corevalve and 78 edwards sapien valves placed in degenerated bioprosthetic valves for aortic stenosis and/or regurgitation . Adverse procedural outcomes included initial device malposition in 15% of cases and ostial coronary obstruction in 4% of cases . Results from a series of 27 cases in germany suggest that transfemoral valve - in - valve implantation, regardless of the failure mode of the original valve, is a feasible and safe option even in older, higher risk patients . Similar results from smaller numbers of patients from another site in germany and from israel have also been reported . We report the first transcatheter corevalve valve - in - valve implantation in a stentless porcine aortic valve in the united states for severe aortic regurgitation . In 1998, a 47-year - old man developed vague symptoms of shortness of breath and fever while on military service in kuwait . He was diagnosed with and treated for bronchitis at the time . When he returned to the united states, he was evaluated with a transthoracic echocardiogram, which revealed mild aortic regurgitation . He was presumed to have had endocarditis and he was treated and followed up . In 2000, his symptoms worsened and he underwent aortic valve replacement with a st ., he refused a mechanical valve prosthesis since he wished to avoid anticoagulation so he could continue active military duty . Shortly after the procedure, his symptoms resolved and then he was lost to follow - up . He presented twelve years later in the fall of 2012, at the age of 59, complaining of progressive exertional dyspnea and chest pain . Despite these symptoms, he was still able to exercise about two hours daily, but he noted that this was a substantial decline from his baseline . In december of 2012, he underwent repeat transthoracic echocardiogram, which showed a moderately thickened and calcified bioprosthetic aortic valve with severe regurgitation . Repeat transthoracic echocardiogram in april of 2013 showed a markedly dilated left ventricle with severe global hypokinesis and a left ventricular ejection fraction of 20% . He was gradually transitioned to his oral regimen of lisinopril, furosemide, and carvedilol . However, his severe physical decline and cardiomyopathy prompted consideration of heart transplant rather than redo aortic valve replacement . He was transferred to our institution and evaluated by the heart transplant team, who deemed him an appropriate candidate for transplant . However, before listing, they consulted the interventional cardiology and cardiac surgery team regarding other possible options . Although it was unclear whether his left ventricular function would recover even after successful valve - in - valve tavr, the interventional cardiology and cardiac surgery team counseled the patient regarding the possible risks and benefits . With the patient's consent pre - tavr evaluation included a coronary angiogram, right heart catheterization, and ct of the chest / abdomen / pelvis . Right heart catheterization showed a right atrial pressure of 6 mmhg, right ventricular pressure of 50/6 mmhg, and pulmonary arterial pressure of 50/26 mmhg with a mean of 34 mmhg . The pulmonary capillary wedge pressure (pcwp) was 27 mmhg, with a blood pressure of 90/50 mmhg, the cardiac output was 3.2 l / min and the cardiac index was 1.9 . The pulmonary vascular resistance was 2.2 woods units and the systemic vascular resistance was 1700 dyn - s / cm . Ct showed an aortic annulus of 20.4 29.2 mm with a perimeter of 76.6 mm and an angle of 35.5 (fig.2). The sinuses of valsalva measured 32.5, 33.5, and 35.3 mm at the noncoronary, left, and right coronary cusps, respectively . The ascending aorta measured 36.8 38.8 mm at a level 40 mm above the valve . The inner diameter of the previous stentless valve was 18.2 24.1 mm with an inner circumference of 67.4 mm . The distance from the valve to the right coronary ostium was 16 mm, and the distance from the valve to the left coronary ostium was 14.7 mm . Ilio - femoral arterial dimensions bilaterally were all greater than 8.3 mm, without significant calcification or tortuosity . Based on these measurements, a femoral approach was planned with the anticipated use of either a 26 or 29 mm corevalve prosthesis . Ct image of pre - tavr stentless aortic valve in cross section . For the procedure, an 18f sheath was inserted via surgical cut down in the right femoral artery and the aortic valve was crossed with a 0.35 mm straight wire . A pigtail was advanced over the wire into the left ventricle and baseline hemodynamic measurements were performed . An exchange length pre - shaped lunderquist wire was advanced against the left ventricular apex . After removal of the pigtail catheter, a 26 mm corevalve prosthesis was passed and partially deployed (fig.3). Even with partial deployment, we found that the annulus of the previous stentless valve gripped the corevalve device . Any attempt to position the device by pulling back encountered considerable resistance . During this process of repositioning, the partially deployed corevalve, we resheathed the device and removed it . Angiographic image of wire / valve angle during first unsuccessful attempt at deployment . Partial deployment of the corevalve is demonstrated, with a relatively vertical angle that leans the corevalve against the lesser curvature of the aortic arch . We next attempted to deploy a 29 mm corevalve prosthesis with the plan to deploy the valve at a greater depth because of the asymmetry of the coronary cusps due to the previously described left cusp . Again when trying to adjust the depth of the device by pulling back on the valve, we encountered considerable resistance, with subsequent popout of the valve again . We resheathed the valve and removed it . Finally, a third corevalve prosthesis (26 mm) was attempted and successful deployment was obtained through a different strategy . Instead of allowing the valve to advance across the annulus with subsequent withdrawal to an optimal position, we pushed the wire against the lv apex, and oriented the device using the left coronary cusp as the reference . Pushing on the wire resulted in its being oriented against the greater curvature of the aorta (fig.4). Following partial deployment, we noted that the device was relatively deep with respect to the non- and right coronary cusps, but was ideally oriented to the deformed left coronary cusp . As opposed to the first two attempts, this time we simply pushed on the wire and did not pull back on the valve with our usual counter - traction . This maneuver resulted in a more optimal angle across the aortic valve, which better matched the orientation of the stentless valve and allowed for successful deployment (figs . 5 and 6). The patient had a rapid postprocedure recovery and reported much improved symptoms by the time of discharge . Discharge tte showed mild - moderate perivalvular aortic regurgitation (fig.7) and follow - up transthoracic echocardiogram at five months showed a well - seated valve with mild perivalvular aortic regurgitation and complete resolution of his lv function to normal . Partial deployment of the corevalve is demonstrated, with a more horizontal angle than the previous attempt, resulting in the corevalve leaning more toward the greater curvature of the aortic arch . Transthoracic echocardiogram demonstrating mild - moderate perivalvular leak s / p valve - in - valve deployment . Our first experience with a stentless porcine valve - in - valve tavr demonstrated many key issues that are crucial to procedural success: (1) the importance of sizing for a stentless valve - in - valve procedure, (2) a different wire technique required in order to successfully deploy a valve - in - valve in the setting of a difficult angle and unusual valve anatomy, and (3) awareness of the difficulty in pulling back the corevalve prosthesis into position through a previous stentless valve . Based on the guidelines for sizing from medtronic, an aortic annulus diameter of 2023 mm should require a 26 mm corevalve prosthesis, an aortic annulus diameter of 2327 mm should require a 29 mm corevalve and a 26 - 29 mm aortic annulus diameter should require a 31 mm corevalve . Based on our patient's aortic annulus perimeter of 76.6 mm, which corresponds to an average diameter of 24.4 mm, the appropriate size corevalve prosthesis should be a 26 mm valve . However, the actual valve dimensions are more elliptical than circular, measuring 20.4 29.2 mm . Therefore, we considered that with one dimension as long as 29.2 mm, it might be preferable to use a 29 mm corevalve . While we were ultimately successful in deploying a 26 mm valve, we believe that our success in the end was likely due to a change in wire technique rather than a difference in valve sizing . Further experience will be helpful in making optimal sizing decisions . Of note, our follow - up echo showed mild aortic insufficiency; it is possible this may have been decreased with the use of a 29 mm valve . While we have previously deployed numerous valves with very horizontal angles (not uncommonly 50 or more), and although this valve only had an angle of 35.5 per the ct scan, the angle along with the partially amputated left coronary cusp posed a more substantial problem than in any of our prior procedures . Our usual technique for positioning the corevalve is to push the stiff wire against the lv apex, while pulling back with counter - traction on the corevalve to provide optimal final positioning after partial deployment . When we tried this method with both the 26 mm and 29 mm corevalve prostheses initially, it did not work . When we finally took the approach of pushing the stiff wire against the lv apex without pulling back on the corevalve, this maneuver allowed the corevalve to lean closer to the greater curvature of the aortic arch, thus sitting more horizontally inside the stentless valve for optimal alignment . A final key lesson for us was that the usual smooth pullback of the corevalve once partially deployed does not work as well when pulling back through a stentless valve . Once partially deployed, the annulus of the prosthetic valve grips the corevalve device, making repositioning very difficult . On our first attempt, when we pulled back the corevalve gently across the stentless valve, it unexpectedly popped back across the valve . When we attempted again with a larger prosthesis, after partial deployment, it caught on the stentless valve, making it difficult to pullback . Ultimately, enough pulling caused it to pop back across the valve rather than sliding back gradually for completion of deployment . During our last attempt, before partial deployment, we carefully positioned the corevalve with our new wire technique for optimal axis orientation, being careful to not start too low to avoid needing to pull back across the stentless valve . This maneuver successfully allowed for full deployment at the appropriate angle and height without requiring any pullback . Successful completion of this procedure supports the idea that stentless valve - in - valve tavr can be a feasible alternative to redo aortic valve surgery in a high- risk patient, if the technical aspects of the procedure can be overcome . In this setting, we recommend careful attention to corevalve size choice, revised aggressive wire technique for angle optimization, and careful initial positioning to avoid the need for repositioning once partially deployed . Based on the guidelines for sizing from medtronic, an aortic annulus diameter of 2023 mm should require a 26 mm corevalve prosthesis, an aortic annulus diameter of 2327 mm should require a 29 mm corevalve and a 26 - 29 mm aortic annulus diameter should require a 31 mm corevalve . Based on our patient's aortic annulus perimeter of 76.6 mm, which corresponds to an average diameter of 24.4 mm, the appropriate size corevalve prosthesis should be a 26 mm valve . However, the actual valve dimensions are more elliptical than circular, measuring 20.4 29.2 mm . Therefore, we considered that with one dimension as long as 29.2 mm, it might be preferable to use a 29 mm corevalve . While we were ultimately successful in deploying a 26 mm valve, we believe that our success in the end was likely due to a change in wire technique rather than a difference in valve sizing . Our follow - up echo showed mild aortic insufficiency; it is possible this may have been decreased with the use of a 29 mm valve . While we have previously deployed numerous valves with very horizontal angles (not uncommonly 50 or more), and although this valve only had an angle of 35.5 per the ct scan, the angle along with the partially amputated left coronary cusp posed a more substantial problem than in any of our prior procedures . Our usual technique for positioning the corevalve is to push the stiff wire against the lv apex, while pulling back with counter - traction on the corevalve to provide optimal final positioning after partial deployment . When we tried this method with both the 26 mm and 29 mm corevalve prostheses initially, it did not work . When we finally took the approach of pushing the stiff wire against the lv apex without pulling back on the corevalve, this maneuver allowed the corevalve to lean closer to the greater curvature of the aortic arch, thus sitting more horizontally inside the stentless valve for optimal alignment . A final key lesson for us was that the usual smooth pullback of the corevalve once partially deployed does not work as well when pulling back through a stentless valve . Once partially deployed, the annulus of the prosthetic valve grips the corevalve device, making repositioning very difficult . On our first attempt, when we pulled back the corevalve gently across the stentless valve, it unexpectedly popped back across the valve . When we attempted again with a larger prosthesis, after partial deployment, it caught on the stentless valve, making it difficult to pullback . Ultimately, enough pulling caused it to pop back across the valve rather than sliding back gradually for completion of deployment . During our last attempt, before partial deployment, we carefully positioned the corevalve with our new wire technique for optimal axis orientation, being careful to not start too low to avoid needing to pull back across the stentless valve . This maneuver successfully allowed for full deployment at the appropriate angle and height without requiring any pullback . Successful completion of this procedure supports the idea that stentless valve - in - valve tavr can be a feasible alternative to redo aortic valve surgery in a high- risk patient, if the technical aspects of the procedure can be overcome . In this setting, we recommend careful attention to corevalve size choice, revised aggressive wire technique for angle optimization, and careful initial positioning to avoid the need for repositioning once partially deployed.
Briefly, we retrospectively reviewed adult mdr tb and xdr tb patients admitted during 20032008 for treatment initiation to king george v hospital (kgvh), a public tb - referral hospital in kwazulu - natal . Admitting physicians and staff collected data routinely . During the study period, kgvh was the only public hospital in the province authorized to initiate treatment for mdr tb and xdr tb, and all therapy was initiated on an inpatient basis . All patients> 18 years of age who had culture - confirmed mdr tb or xdr tb with standard drug susceptibility testing were included . Ethics review committees at the university of kwazulu - natal and boston university (boston, ma, usa) approved the study protocol . Univariate and multivariate logistic regression models were used to estimate odds ratios (ors) and 95% confidence intervals (cis). Statistically significant variables or variables that caused> 10% change in the univariate or were included in the multivariate model . A total of 4,941 patients with mdr tb or xdr tb from throughout kwazulu - natal were admitted for initiation of drug - resistant tb treatment to kgvh during the study period (figure). Among 4,514 eligible patients with mdr tb or xdr tb, women were younger (median age [interquartile range] 32 [2639] vs. 36 [3044] years), more likely to be hiv infected (65% vs. 47%), and more likely than men to be receiving antiretroviral therapy (art) (51% vs. 43%) (table 1). Women with drug - resistant tb were more likely than men with drug - resistant tb to have xdr tb (p<0.0001). Flow diagram for patients with multidrug - resistant (mdr) and extensively drug - resistant (xdr) tuberculosis (tb) admitted to king george v hospital, kwazulu - natal, south africa, 20032008 . * mdr, multidrug - resistant; tb, tuberculosis; xdr, extensively drug - resistant; art, antiretroviral therapy . Median patient age (interquartile range): women, 32 y (2639 y); men, 36 y (3044 y); p<0.0001 . On univariate analysis, hiv status, art (among hiv - infected), female gender, previous tb treatment, and year of admission to kgvh were significantly associated with xdr tb (table 2). On multivariate analysis, only female gender (or 1.38, 95% ci 1.111.73), previous tb treatment (or 2.16, 95% ci 1.094.28), and year of admission were independently associated with xdr tb . In the hiv strata, art was not significantly associated with xdr tb after adjustment was made for confounding variables . Confounding of art and xdr tb by year of admission among hiv co - infected persons most likely resulted from increased xdr tb case finding after a highly publicized xdr tb outbreak in 2006 and increased art use after public health rollout of art in kwazulu - natal in 2004 . The interaction terms hiv gender, gender age (categorical), and health care worker (hcw) gender were not significantly associated with xdr tb . * xdr, extensively drug - resistant; tb, tuberculosis; mdr, multidrug - resistant; or, odds ratio; ci, confidence interval; art, antiretroviral therapy . Median age (interquartile range): mdr tb patients 34 y (2842 y); xdr tb patients, 35 y (2942 y). Most (59%) patients admitted with xdr tb were women, which did not change significantly during the study period (test for trend p = 0.68). For mdr tb, the data showed increasingly more female mdr tb patients admitted over the study period (p<0.001) (table a1). Our major finding was that women admitted with drug - resistant tb to kgvh were 38% more likely than men to have xdr tb . Temporal analysis showed persistently more women with xdr tb and increasing proportions of women with mdr tb during the study period . Together these data support the notion that the epidemic of drug - resistant tb predominantly affects women in kwazulu - natal . Supporting context comes from observational studies of drug - resistant tb in south africa . In south african studies, higher percentages of xdr tb (50%56%) than mdr tb patients (43%53%) are women (69). However, studies from low - prevalence hiv settings report fewer women with drug - resistant tb . In a study in the united states, few patients with mdr tb (36%) or xdr tb (38%) were female (11). Similarly, in cohorts from latvia, peru, and russia, lower percentages of patients with mdr tb (17%40%) and xdr tb (29%35%) were female (1214). Gender differences in drug - resistant tb in areas of hiv endemicity and low prevalence suggest a possible effect of the aids epidemic on prevalence of drug - resistant tb in women . Women with xdr tb might have been preferentially referred compared with men with xdr tb, women with mdr tb, or both . We lacked data on hiv factors, such as cd4 t - cell counts, art adherence, and viral load . Similarly, we lacked data on previous tb treatment, medications, adherence, and outcome . Finally, as a hospital - based retrospective study, factors associated with survival to hospital admission, decisions to seek care, and referral patterns may introduce bias . For example, women with drug - resistant tb may be more adherent to art, leading to improved survival, and therefore increased time for xdr tb to develop . Factors associated with secondary development of xdr tb, such as tb medication adherence or previous mdr tb treatment, could explain the association between xdr tb and female gender (15). Factors associated with exposure to drug - resistant tb strains, including location and duration of exposure, could explain the association of xdr tb and female gender . For example, women are more likely to participate in formal and informal care work, with potential exposure to drug - resistant tb strains, and therefore primary xdr tb might be more likely to develop (5). These results may have major policy implications for tb control in kwazulu - natal and south africa . Because women are more likely to have xdr tb in kwazulu - natal, efforts should be made to develop gender - sensitive interventions to improve diagnosis, treatment, and prevention for drug - resistant tb and hiv . Decentralization of drug - resistant tb treatment may better accommodate women in conjunction with their work, family, and child - rearing responsibilities . Further studies are needed to confirm the magnitude and determinants of the association between female gender and xdr tb.
We analysed data from the czech part of the hapiee project (peasey et al . The study recruited over 8000 caucasian men and women aged 4569 years at baseline (20022005), randomly sampled from population registers of seven czech towns . Dna was extracted from venous blood samples and apoe genotypes were determined using the conventional pcr - rflp method (hixson & vernier, 1990). Two criteria were adopted to define hand dominance, both using data collected during the re - examination of the cohort in 20062008 (total of 5362 participants). Second, we used grip strength data and classified as left - handed those who recorded higher grip strength on their left hand on both successive tests . Both hand dominance and apoe genotype were available for 4438 participants (2022 males and 2416 females), mean age 58.0 (sd 7.1) years . Differences in the proportion of left - handed individuals between apoe subgroups, both in the full sample and when stratified by age and sex, were assessed by a chi - square test . In addition we tested the association between left - handedness and the presence of the apoe2 allele vs either apoe33 or 43/44, using logistic regression . We also assessed the association separately by 5-year age group, and we tested for potential interaction between age group and apoe genotype . All analyses were conducted using the stata version 12 statistical software (station college, tx, usa). The study was approved by the local ethics committees at both czech national institute of public health and at university college london . A total of 217 (4.9%) individuals reported having a dominant left hand for writing and 543 (12.2%) persons had consistently higher grip strength on their left hand . These two measures were strongly correlated; individuals with higher left hand grip strength were five times more likely to be self - reported left - handers (odds ratio 5.2, 95% confidence interval 4.06.7, p <.0001). The frequency of the most common apoe3 allele was 81.1%, followed by the apoe4 allele (11.3%), and the least common was the apoe2 allele (7.5%). The distribution of left - handers based on either self - report or grip strength did not differ between carriers of different apoe genotypes; the p - values were 0.828 (2.1, 5 df) and 0.557 (3.9, 5 df) (table 1). Similarly, there were no statistically significant differences when carriers of apoe2e2 and apoe2e3 were combined, and compared with apoe3e3 homozygotes and with apoe4 (apoe4e3 and apoe4e4) carriers, with no odds ratio being anywhere close of statistical significance (table 2). Distribution of left - handers among the different apoe genotypes age - sex - adjusted and age - specific odds ratios of left - handedness by presence of apoe2 allele, with confidence intervals and p - values . Left - handedness was the dependent variables coded as 0 (non - left - handed) and 1 (left - handed); the presence of apoe2 allele was coded as 0 (no apoe2 allele present) and 1 (at least one apoe2 allele present). Adjusted for age and sex further analyses, stratified by 5-year age group, did not reveal any significant association between left - handedness and apoe genotype in any age group, nor any interactions between apoe and age group (table 2). Similar analyses, stratified for sex, also produced negative results (table 2). While many studies have suggested a genetic contribution to handedness (see annet, 1994), very few studies investigated specific genetic markers in relation to left-/right - handedness (scerri et al . This study, a study of children, did find an association between apoe and left handedness (bloss et al ., 2010). Our study, much larger than the previous investigation but in adults, did not find any association between apoe genotype and hand dominance . The apoe genotype frequencies in our study were similar to the frequencies detected in neighbouring populations (bazrgar et al . 2008; gerdes, klausen, sihn, & faergeman, 1992); they also did not differ significantly from the study by bloss et al . The genotype frequencies were also similar to a study conducted in neighbouring slavonic / polish population in which the apoe4 allele was associated with elevated risk of alzheimer disease (klimkowicz - mrowiec et al ., 2010). There are two main limitations of our study: the measurement of handedness and the restricted age range of the examined individuals . Both these factors limited our ability to analyse life - long handedness and possible redirection of handedness in childhood . Hand preference is more a continuous rather than binary trait, and ambidexterity may be more common than strict left - handedness (llaurens et al ., the assessment of handedness in our study was based on subjective self - report and on measurement of grip strength, not allowing to us to detect the potential ambidextrous individuals . None of our two outcome classifications is ideal, especially because the age groups included in our study might have been forced to use their right hand when they were children . This fact may also explain the discrepancy between self - report and grip strength results, although in both methods the proportions of left - handers were reasonably close to the expected range of 5 to 10% (llaurens et al ., 2009). On the other hand one might argue that the degree of enforcement might have differed between sexes or between birth cohorts; the absence of any confounding or effect modification by age or sex contradicts the presence of a major bias . The observations that the proportion of left - handed persons based on grip strength was close to prevalence of 12% reported in an earlier study in the czech population (dvorakova, & zvolsky, 1989), and the expected higher prevalence in males than females, further suggest that the grip strength based measure may be the more reliable of the two measures . Our study had sufficient power to detect relatively modest differences in the prevalence of left - handedness between apoe2 carriers and carriers of others genotypes, and the three - fold difference reported by bloss et al . However, we cannot completely exclude the possibility that small differences exist in left - handedness and apoe genotypes . Given the low prevalence of apoe2e2 and apoe4e4 homozygosity (less than 1%), a much larger study would be needed to demonstrate such a small effect for these rare genotypes . Notwithstanding these limitations, there was no suggestion of an association between apoe genotype and left - handedness . (in press). We therefore conclude that common polymorphism within the apoe gene is likely not a major genetic determinant of left - handedness in adults.
Diaphyseal aclasis, also termed hereditary multiple exostosis (hme) or osteochondromatosis is characterised by multiple bony prominences that grow near joint lines throughout the skeleton . These bony prominences, otherwise known as exostoses, can present as painless bony deformities or as a complication of the bony growth . (1) there have been approximately 100 cases in the literature of vascular complications due to osteochondromas including pseudoaneurysm formation, occlusion, rupture or thrombosis . (2) this article describes the case of a 15 year old boy, with known hme, referred to the sarcoma mdt with a 9.5 cm diameter mass in the popliteal fossa, confirmed by ultrasound as a delayed presentation of a pseudoaneurysm . A 15-year - old boy, with diaphyseal aclasis, presented with a two month history of a painful swelling in his left popliteal fossa, reportedly increasing in size . He presented to his local orthopaedic department where repeated radiographs (figure 1) illustrated decreased cortical definition of the exostosis arising from the medial femoral metaphysis 15 cm above the knee joint . At this point he was referred to the regional sarcoma mdt due to concern that the lesion had undergone malignant transformation . Plain radiographs of the patient s left knee on examination, there was a 10 cm, non - tender, pulsatile mass in the popliteal fossa . The foot was warm with normal capillary refill, palpable posterior tibialis artery but absent dorsalis pedis . The mri scan showed a lesion measuring 9.5 cm in its greatest diameter, of uniform signal, with a cartilage cap exceeding 2 cm throughout most of its surface . (figure 2) mri scan of the patient s left leg an ultrasound concluded an 8.8 cm pseudoaneurysm and the patient was admitted for urgent pre - op ct angio (figure 3 and 4) and surgical exploration . Ct angiogram 3d reconstruction- showing the bony exostoses and the popliteal aneurysm posterior to the right distal femur ct angiogram: showing the diameter size of the aneurysm to be 88.6 mm . The cross sectional image clearly shows the sharp bony exostoses that slowly eroded into the popliteal artery resulting in the pseudoaneurysm formation . The image also shows that the defect in the artery is still patent because the pseudoaneurysm is filling with contrast the popliteal artery was explored . (figure 5) the proximal popliteal artery was found to have a 1 cm x 0.5 cm wall defect adjacent to the bony exostosis . (figure 6)the exostosis was excised and the 4 cm of diseased artery repaired with end - to - end interpositional long saphenous vein graft . (figure 7) intraoperative photograph showing the popliteal aneurysm intraoperative photograph showing the 1 cm x 0.5 cm defect in the popliteal artery wall caused by the sharp bony exostoses intraoperative photograph showing the end to end interpositional long saphenous vein graft post operatively the patient recovered without complication . He was followed up with an ultrasound doppler after 6 weeks demonstrating a patent vein graft and will undergo local vascular follow - up . His 6 month post - operative check - up revealed no complications but he will have regular follow - up for any painful, rapidly growing exostosis associated with his hereditary condition . Diaphyseal aclasis is a rare genetic skeletal condition due to developmental abnormalities of the growth plate causing multiple cartilage covered exostoses to form on the surface of the metaphysis or the adjacent diaphysis region of long bones . Diaphyseal aclasis is usually diagnosed in childhood however it is believed that radiologically the exostoses are present since birth . (3) vascular complications of the popliteal vessel occur due to ossification of the previously protective cartilage cap, around the 2 decade and relative immobility of the artery in the popliteal fossa between exiting hunter s canal and the trifurcation . (4) the artery undergoes chronic abrasion on the sharp exostosis forming a defect in the adventitia resulting in pseudoaneurysm formation . (5) it is controversial whether exostoses that lie near a vascular axis should be removed on identification to prevent vascular compromise from occurring . Considering most exostoses are innocent, most authors suggest that surgical excision is only required when complications have occurred or malignant transformation is suspected . If preventative surgery is not performed, regular ultrasound doppler scans should be performed in order to screen for complications . (6) in addition to vascular complications, bony exostosis can also present acutely with nerve compression, bursitis due to bony irritation, stalk fracture or necrosis of the cartilaginous cap following infarction . (7) due to the acute presentation of this vascular complication there was no time for pre - operative tissue diagnosis, which would be routinely performed . It was important that the procedure was performed by an experienced orthopaedic tumour surgeon to ensure adequate margins were achieved . Recent studies report a lifetime risk of sarcomatous transformation in single lesions of approximately 1% but up to 5% in hme . (8) the majority are chondrosarcomas, however, osteosarcomas and malignant fibrous histiocytomas have been reported . Concerning features that warrant further investigation include: a rapidly enlarging mass, particularly in those beyond skeletal maturityshoulder girdle and pelvic exostosis (9)cartilage capsule exceeding 1.5 cm a rapidly enlarging mass, particularly in those beyond skeletal maturity shoulder girdle and pelvic exostosis (9) cartilage capsule exceeding 1.5 cm investigation with ultrasound, mri and image guided or open biopsy is advocated if an exostosis has concerning features . There are recent studies advocating pet / ct imaging to monitor known benign exostosis for malignant transformation . (10) due to the relative rarity of these lesions it is important to review the images and histology in a specialised bone tumour multi - disciplinary meeting . This case illustrates a late presentation of a known complication associated with diaphseal aclasis . In this age group we present one of the largest, non - ruptured, popliteal pseudo aneurysms reported . It is important to maintain a high index of suspicion of an increasing soft tissue lesion due to the risk of sarcomatous transformation in patients with mhe; however, this focus delayed earlier presentation to vascular surgeons putting the patient at risk of a limb threatening rupture.
A child is brought into a pediatric emergency unit with an unprovoked, afebrile first seizure . We conduct a clinical assessment of the child and rule out any acute metabolic, traumatic or infectious causes and consequently, make the diagnosis of an epileptic seizure . The international league against epilepsy suggests that following such a diagnosis, the next step should be the appropriate classification of the seizure type, after which an appropriate syndrome diagnosis should be made . Should an electroencephalogram (eeg) be arranged for this child and if so, should it be arranged within 24 h or within the next week? If we decide not to arrange an eeg this time and to do so if any further seizures occur, are we practicing evidence based medicine? A recent guideline published by the royal college of pediatrics and child health (rcpch) asserted: there is no need for an eeg following a first simple afebrile seizure . This is a very bold and clear statement however, what evidence and what quality of evidence is this statement based upon? This review analyses and discusses prominent literature regarding this widely - discussed topic . Whilst literature on this controversial subject matter is very diverse, a large proportion of the debate surrounds the utility of the eeg for the counselling of parents / family following a first seizure; particularly as to whether it is a viable tool to predict seizure recurrence . One study conducted by stroink et al . Aimed to assess the accuracy of the diagnosis of a first unprovoked seizure in childhood, the recurrence rate within 2 years, the risk factors for recurrence and the long - term outcome 2 years after recurrence . A panel of three neurologists confirmed the diagnosis of seizure in children based on pre - described diagnostic criteria . All the children deemed as having a seizure had a standard eeg, if it did not disclose an epileptiform abnormality, a second eeg was performed after partial sleep deprivation, or during daytime sleep . One of the most interesting results was the recurrence rate of 71% in children who had an epileptiform eegs in standard eeg . They concluded by stating that an eeg with epileptiform abnormalities proved to be the main risk factor for recurrence . However, we must consider whether having this information after the first seizure would change our practice; it must be noted that a significant proportion (29%) of children with epileptiform eegs did not have a recurrence . The authors, in their prospective cohort study on 347 children with a first, unprovoked afebrile seizure showed that children with an idiopathic first seizure who had an abnormal eeg were at an increased risk of seizure recurrence . 54% of those with epileptiform eegs experienced a second seizure, in contrast to 25% recurrences in those who exhibited a normal eeg . They concluded that an abnormal eeg, particularly an epileptiform abnormality, is associated with a higher risk of seizure recurrence in children with an idiopathic first seizure . Whilst shinnar et al ., have clearly demonstrated the higher risk of seizure recurrence in children with interictal eeg abnormalities, it is essential that we remember that a significant proportion of children with an abnormal eeg had no seizure recurrence . Although, an abnormal eeg would increase the risk of recurrence by nearly two folds, whether this has to be included in counseling patients and families is debatable, as an abnormal eeg is not an absolute predictor of seizure recurrence, and a normal eeg may be falsely reassuring . A review of literature conducted by pohlmann - eden et al . On the use of the eeg and neuro - imaging following an epileptic seizure also focused on the potential interpretations and limitations of the eeg following a first seizure . They reviewed literature (children and adults) on seizure recurrence, timing of eeg, yield of abnormalities on eeg, yield and timing of sleep deprived eeg and focal epileptiform activity in relation to recurrence . They concluded that an early abnormal eeg especially, when showing focal epileptiform activity seems to be an excellent predictor for seizure recurrence . Would a physician be able to effectively counsel children and carers based on the percentages identified in the studies appraised above and more importantly, is having a recurrence rate for any type of seizure contributory to the management or counselling of patients? Another argument put forward by advocates of the use of eeg - after first seizure is its application as a prognostic marker . Dr panayiotopolous, from the department of clinical neurophysiology and epilepsy at st . Thomass hospital, recommended that a revision of the current practice in pediatrics of not requesting an eeg after the first afebrile seizure may be needed . He argued that the current practice of treating after two seizures is not in keeping with the best - available evidence . He commented that an abnormal eeg, particularly generalized spike wave discharges have been reported as a consistent predictor of recurrence in nearly all studies . He discussed the benefits of having an eeg after first seizure; these include diagnosis of a specific epileptic syndrome, making a diagnosis of benign childhood partial seizures (as these children may not have more than a single episode) and also assessing the diagnostic and management applications of minor seizures (such as absences and myoclonic jerks). He clearly defined the advantages of an eeg after the first seizure, stating that the child and the family are entitled to a diagnosis, prognosis and management that is specific and precise, even though this may only be possible in a select - proportion of patients after the first seizure . In his recommendations, panayiotopoulos also commented that the eeg was a harmless investigation; this point brought disagreement from pediatric neurologist, richard appleton . He emphasized that although the eeg was harmless with regards to its invasiveness, it could be potentially harmful in terms of its interpretation . Due to the variability in the quality of eeg reports and interpretation, there may be a significant number of inaccurate diagnoses, which could consequently lead to serious medical, psychological and social consequences . He also added that irrespective of the eeg changes, many children would not be treated after a first seizure owing to the high non - recurrence rates after first seizures . He has also pointed towards the fact that most children are managed outside tertiary epilepsy centers and may not have access to medical personnel who should be undertaking and reporting eegs . The author has clearly highlighted very important issues that would be of immense practical importance and would also initiate thinking in these under - emphasized areas of this contentious topic . Some of the pitfalls of pediatric eeg interpretations were also discussed by tan et al . ; their article was written in response to the clinical guidelines published by the national institute of clinical excellence (nice) in 2004 . They stated that the eeg has rarely been able to function as a black or white or yes or no diagnostic test for epilepsy, whilst also commenting that the normal eeg evolves and matures with time, reaching the normal adult pattern by approximately 12 years of age . They proceeded to describe eeg reading as an inexact science, which is learnt through experience and stated that the accuracy of eeg interpretation relies heavily on the clinical history provided by the physician, which on many occasions is insufficient . They discuss the fact that in the united kingdom, eegs are reported by different medical practitioners including neurophysiologists, neurologists (both pediatric and adult), psychiatrists and even general practitioners with a special interest in the eeg . Many of these practitioners will have had very little exposure to children and pediatric epilepsy, thus, it is difficult to monitor the quality of eeg reporting . The authors have clearly highlighted the varied quality of eeg reports in various settings and therefore, this has to be considered when using the eeg to guide management of an individual case, especially, if the eeg has been acquired after a first unprovoked seizure . Another argument that has been put forward against the routine use of the eeg following the first seizure is whether or not it will contribute to diagnosis or management . Conducted a medline search from 1980 to 1998 to quantify and analyze the value of expected information from an eeg after first unprovoked seizure in childhood . They employed an evidence - based analytical approach to published literature to select studies providing reasonable estimates of the value of eeg in the general population of children with first unprovoked seizure . Next, the information from these studies was quantified using linear information theory and their results showed that the likelihood of making a clinically useful diagnosis by performing an eeg in every child after first seizure was low . In addition to this, they commented that as a result of the low sensitivity and specificity of the eeg, many false predictions could occur, thus, resulting in unnecessary patient distress or reassurance . The quality of expected information from the eeg is too low to affect treatment recommendations in most patients, thus, they concluded by stating, eeg should be ordered selectively, not routinely, after first unprovoked seizure in childhood . Their conclusion was countered by berg et al . Who stated that gilbert et al . Focused on the impact of eeg on treatment decisions and neglected much of the additional value of an eeg . He argued that an eeg following the first seizure may provide valuable diagnostic and prognostic information despite epilepsy being a clinical diagnosis . Furthermore, an abnormal eeg in an appropriate clinical context may help support the diagnosis of a seizure disorder, whilst also helping to distinguish between partial, and generalized seizures . In their study, gilbert and on the utility of eeg for diagnosis following first seizure, which stated that differentiating partial from generalized seizures was non - specific; berg et al . Argued that this non - specific distinction has fundamental treatment implications if recurrent seizure occur, as the first line anti - epileptic drugs are different for partial and generalized epilepsy. [911] however, it is important to note that although differentiating partial and generalized epilepsy may have treatment implications (it is not common practice to treat after first seizures) these are not specific syndromic diagnoses and thus, having this information may not contribute to the first counseling the child or carers receive . Both nice and scottish intercollegiate guidelines network in their guidelines acknowledged that epilepsy is primarily a clinical diagnosis and that, in isolation, the eeg should not be used to make a diagnosis of epilepsy . At present, the majority of pediatricians do not request an eeg after a first seizure and this practice has been emphasized at a recent rcpch guideline appraisal . Eeg carried out at this point has a low sensitivity and specificity for recurrence rates, which may lead to false predictions . Therefore, the information on recurrence may not be helpful in counseling; however, an abnormal eeg would increase the risk of recurrence by nearly two folds . There are also a significant proportion of children with first unprovoked seizure and abnormal eeg who may not have any further seizures . Therefore, including the information in counseling may lead to unnecessary psychological stress for both children and carers as the finding of an abnormal eeg does not imply that the child will invariably have a further seizure . Most parents would opt against giving their children anti - epileptic medications after a first seizure, provided they are given the right information that it is possible that there may be no recurrence . The other aspect to consider is the quality of eeg interpretation that a physician receives to base his judgment on and as mentioned above, quality control of the eeg is very difficult to regulate . On the other hand, there are several clinical situations in which conducting an eeg after a single seizure can lead to the diagnosis of specific epileptic syndrome . Secondly, minor seizures such as absences and myoclonic jerks could be picked up, both for which prognostic and treatment implications could be deduced . Children and their carers are entitled to a specific diagnosis, a prognosis, and a precise management plan . Diagnosis of a seizure, epilepsy type and an epileptic syndrome should be primarily based on clinical grounds . If it is requested, then it should be to answer a specific question, which in turn would help to aid the diagnosis of epilepsy . The quality of the eeg report and its interpretation has to be considered, and if in doubt, further specialized advice should be sought . As there is limited literature on eegs after first seizure in varying age groups, conducting a prospective study to evaluate the eeg after first seizure is needed . In particular, to assess the influence of the eeg results on treatment decisions, social and emotional implications and seizure recurrence . Diagnosis of a seizure, epilepsy type and an epileptic syndrome should be primarily based on clinical grounds . If it is requested, then it should be to answer a specific question, which in turn would help to aid the diagnosis of epilepsy . The quality of the eeg report and its interpretation has to be considered, and if in doubt, further specialized advice should be sought . As there is limited literature on eegs after first seizure in varying age groups, conducting a prospective study to evaluate the eeg after first seizure is needed . In particular, to assess the influence of the eeg results on treatment decisions, social and emotional implications and seizure recurrence.
Thanks to technological progress, in the last 30 years there has been a significant increase in the number of patients with esrd (end - stage renal disease) undergoing renal replacement therapy . Despite this progress, recent statistics show that technological advancement does not translate into reduced mortality or morbidity in chronic dialysis patients . Yearly mortality in the group of patients with chronic kidney failure in stage 5 d varies from 6.6% in japan, 15.6% in europe, and 21.7% in the usa . Although most therapy - optimizing guidelines were implemented with maximum efficiency, morbidity of hemodialysis patients in the usa decreased by only 1% per decade and the average duration of hospitalization did not fall below 15 days per year . This data indicates the need for early identification of classic risk factors for cardiovascular diseases such as coronary heart disease and chronic heart failure with arrhythmic complications, as recognized predictors of fatal prognosis in dialysis patients with esrd . In recent years, substantial attention was paid to the development of pulmonary hypertension (ph), as another strong risk factor for adverse outcomes in a population of patients on both hemodialysis (hd) and peritoneal dialysis program (pd)., who defined the ph with an associated right ventricle failure as a new challenge for cardionephrologists in the 21st century . Our article presents the usefulness of echocardiography in the early diagnosis of ph in dialysis patients, based on our own clinical experience . Common guidelines of the european society of cardiology (esc) and the european respiratory society (ers) for the diagnosis and treatment of pulmonary hypertension define it as the pathological and hemodynamic state characterized by elevated mean pulmonary artery pressure 25 mmhg at rest diagnosed with right heart catheterization (rhc). Pulmonary hypertension occurs in the course of a number of diseases qualified by esc and ers to five categories covering 37 clinical units of different pathogenesis and clinical course, as well as therapy . Thus, group 1 includes pulmonary arterial hypertension (mainly idiopathic and genetically determined); group 2 ph due to left heart diseases; group 3 ph due to lung diseases; group 4 chronic thromboembolic pulmonary hypertension; group 5 pulmonary hypertension of unclear and/or multifactorial pathogenesis, including chronic kidney disease (ckd). Havlucu et al . In their prospective study in which 211 patients with esrd were enrolled similar results were achieved by abdelwhab and elshinawy, who in the group of 76 patients with esrd recognized pulmonary hypertension in 32% of patients treated conservatively and in 44% of hemodialyzed patients ., analyzing the incidence of echocardiographic signs of right ventricular dysfunction in a population of 120 dialyzed patients demonstrated that those abnormalities occur significantly more often in patients treated with hemodialysis (hd) than peritoneal dialysis (71% vs. 35%). Analyzed the results of 13 observational studies on echocardiographic diagnosis of pulmonary hypertension in esrd patients and concluded that this problem occurred in 3060% patients treated with hd . However, in the population of patients on pd, pulmonary hypertension was diagnosed less frequently in 12 to 42% of patients, which was comparable with the results obtained in a population of ckd patients treated conservatively . In the latter group, ph was diagnosed in 13 to 39% of patients . Regarding patients undergoing hd, creation of proximal arteriovenous fistula and proximal location of avf compared to its distal location might cause blood flow elevated up to 1.01.5 l / min through extracorporeal circulation and consequently, recirculation of blood through the pulmonary vascular bed increase up to 20%, which additionally causes right heart volume overload in hemodialysis patients . According to amerling et al . Hemodynamic consequences of avf responsible for the development of ph are increased blood flow and pressure in the pulmonary circulation, as the consequences of an increase in cardiac output, decrease of peripheral resistance and increased activity of the sympathetic nervous system . The right heart catheterization (rhc) remains a gold standard for the diagnosis of ph, but as an invasive test is available only in specialized clinical centers . In accordance with the guidelines of the european society of cardiology from 2009, ph can be identified when the test shows the average resting mean pulmonary artery pressure 25 mmhg (mpap). Taking into account the non - characteristic clinical manifestation of ph in the form of exertional dyspnea and weakness, before qualifying the patient for rhc, the initial diagnosis of ph should be confirmed by non - invasive diagnostic tests . This restricts the population to the group of patients suspected, in which invasive diagnostic is fully justified . In everyday clinical practice, transthoracic echocardiography (tte) is the most frequently used for this purpose non - invasive diagnostic test . Eugene braunwald, in a lecture delivered on 09/02/2013 at the congress of the european society of cardiology in amsterdam counted echocardiography among ten greatest achievements of modern cardiology . Contemporary echocardiography is becoming available not only in specialized institutes, but also directly at patients bedside . This diagnostic method allows non - invasive assessment of pressure in the pulmonary circulation and imaging of morphological, functional as well as hemodynamic consequences of ph . Finally, it is possible to analyze simultaneously the severity of ph and differentiate its potential causes . According to the cited esc recommendations available modes of echocardiographic imaging, ranging from one - dimensional echocardiography, through two - dimensional, three - dimensional, as well as the doppler techniques, intravascular and intracardiac echocardiography, answer most questions about the structure and function of myocardium, as well as major vessels of the chest . What is important from a practical point of view, the patient does not need to be specially prepared for the tte, which significantly simplifies the process of diagnosis and treatment . It should be noted that computed tomography often preferred to echocardiography is associated with both short - term risk of complications (allergic reactions to the contrast agent, the deterioration of renal function) and long term reactions related to ionizing radiation . In summary, modern echocardiography allows simultaneous estimation of many indicators of right ventricular function and pulmonary circulation and should be carried out as a first - line test in every patient with suspicion of pulmonary hypertension . Doppler assessment of pulmonary artery systolic pressure (pasp) which is synonymous after exclusion of pulmonic stenosis with right ventricular systolic pressure (rvsp) remains the basis of echocardiographic evaluation of pulmonary pressure and right ventricular function . Using the tricuspid regurgitant jet velocity recorded with continuous wave doppler, the bernoulli equation (maximum gradient=4v) is used to obtain the right ventricular to right atrial systolic pressure gradient (tricuspid regurgitation peak gradient, trpg). Trpg value> 31 mmhg (i.e. Tricuspid regurgitation velocity> 2.8 m / s) suggests ph . In turn, the value of rvsp (allowing the estimation of pasp) is obtained by adding to the trpg value of rap (pressure in the right atrium of the heart): the rap estimation is on the diameter and respiratory variability of the inferior vena cava (ivc). Thus, inferior vena cava collapsing about 50% during inspiration with a width less than 15 mm indicates the proper rap (about 5 mm hg). Inferior vena cava with the width reduction 15 mm or without inspiratory collapse calls for rap increase by more than 20 mm hg . If calculated according to the above presented principle rvsp value exceeds 3540 mmhg then patient requires a more detailed assessment towards ph . Due to the relative simplicity of this method in clinical practice the tricuspid regurgitant jet in conjunction with an estimate of right atrial pressure is used in most laboratories to calculate pulmonary artery systolic pressure . Figure 1 shows principle of rvsp assessment using continuous doppler wave apical four - chamber projection . It is worth noting that the only hemodynamic parameter included in the definition of ph is the mean value of pulmonary artery pressure (mpap). Its estimation using pasp value assessed with doppler echocardiography is obtained through a number of formulae . One of them developed by a group of french researchers (chemla d, castelain v, humbert m. et al .) Is as follows: other doppler methods for estimating pulmonary artery pressure include measurements of early- and late - diastolic regurgitant velocity on the level of the pulmonary trunk valve and the time to peak velocity in the pulmonary outflow tract (also called acceleration time act). In the case of the latter parameter shortening of the act<60 msec at moderately elevated pasp (<60 mmhg) is one of the specific echocardiographic indicators of acute pulmonary embolism . The limitations of doppler technique measurement of the pressure in the pulmonary artery are considerable . This test does not directly measure the pressure in the pulmonary arteries and may be inaccurate, particularly in the case of impaired function of the right heart or cardiac arrhythmias . In the first case, the systolic right ventricular dysfunction and elevated right atrial pressure will result in lower systolic gradient between these cavities and underestimation of pulmonary hypertension values . In contrast, atrial fibrillation with a high ventricular rate or other cardiac arrhythmias can cause large fluctuations in the trpg values, which depend on the variable time of the right ventricle filling . The use of the aforementioned method in patients with arrhythmias is associated with frequent overestimation of pasp, occurring in up to 50% of investigated patients . Therefore, according to the esc guidelines for the diagnosis and treatment of pulmonary hypertension echocardiography can only determine the probability of the occurrence of ph, and not its actual severity . Table 2 shows the parameters defining the low, moderate, and high probability of ph based on echocardiography . In every patient with clinically suspected ph, a necessary element of echocardiographic examination is to assess the function of right ventricle and degree of its overload . Suspicion of the ph suggests the presence of increased right ventricular dimensions with flattening, displacement, and paradoxical movement of the interventricular septum (ivs) in the direction of the left ventricle . Thickening of the right ventricle walls and the pulmonary artery trunk extension also suggest ph, but appear in the later stages of this pathology . It should be emphasized that the role of echocardiographic assessment of morphological changes in the diagnosis of ph increases in cases of unreliable pressure measurement with doppler method (e.g. In arrhythmias with high ventricular rate). According to the current guidelines for the assessment of right ventricular systolic function the following echocardiographic parameters should be used: the amplitude of tricuspid annular plane systolic excursion (tapse); the percentage of fractional area change (fac); the measurement of the longitudinal velocity of excursion of the tricuspid annulus and the basal free wall segment of the right ventricle (rv s) with tissue doppler echocardiography (tde). Tricuspid annular plane systolic excursion (tapse), described by kaul in 1984 and introduced to the clinical practice by hammarstrom in 1991, is a measurement of m - mode echocardiography that reflects longitudinal contraction of the right ventricle and is the easiest, well documented, quantitative index of right ventricular function . Samad et al . In their study of 194 patients with acute myocardial infarction found that the value of tapse <15 mm is associated with a significantly higher risk of death (45%) in the two year follow - up compared to patients with tapse> 20 mm (4%). Tapse and fac measurements significantly correlated with the value of the ejection fraction of the right ventricle assessed with magnetic resonance and radioisotope examinations . . It should be noted, however, that the time - consuming measurement of fac makes it of limited clinical utility . Tissue doppler echocardiography (tde), which recently becomes more available, may also be used to assess the systolic function of the right ventricle . The reduced <1012 cm / s maximum speed of tricuspid annular motion (s) is a symptom of right ventricle dysfunction . It should be noted that due to the complicated geometry of right ventricle cavity, the measurement of right ventricular ejection fraction, in contrast to the left ventricle, cannot be applied in everyday practice . Measurement of tapse represents a novelty in the diagnosis and monitoring of ph in dialyzed patients . Implementation of this technique into everyday practice does not require sophisticated equipment or prolonged image analysis and is fairly simple even for less experienced physicians . Another advantage is the fact that tapse can be determined in patients with atrial fibrillation with a high ventricular rate in whom reliable echocardiographic examination, including doppler measurements, may be difficult to perform . Tapse should be measured in the apical four - chamber projection using a one - dimensional echocardiography (m - mode). Tapse value determines the range of motion or displacement of the tricuspid valve ring in the longitudinal plane in successive phases of the cardiac cycle, reflecting the function of the right ventricle . Forfia et al . In a prospective study of 63 patients diagnosed with ph showed that the value of tapse less than 18 mm was an independent risk factor for mortality in this population . Table 3 summarizes the normal values of basic echocardiographic measurements for assessing the structure and function of the right heart . In a group of 202 patients with stage 3 ckd and a negative history of ph discovered reduced values of tapse (<23 mm) in 43% of the studied group . Tapse showed a statistically significant negative correlation with the values of systolic blood pressure in the pulmonary artery . Consequently, statistical analysis enabled isolation of subpopulation of patients at risk of developing pulmonary hypertension . This group was characterized by low values of tapse (around 16 mm) and high values of systolic blood pressure in the pulmonary artery (about 38 mmhg). It should be pointed out, that the study by floccari et al . Included patients in the early stages of ckd, which means not on dialysis . The strength of this study was the identification a subpopulation of patients at risk of developing ph . According to the authors these patients in the case of sudden progression of ckd and need for dialysis should not be qualified to proximal, i.e., high avf but either for a distal avf, or for peritoneal dialysis . The authors noted also that in the case of congestive heart failure, shortness of breath and a decrease in daily diuresis, aside from an effect of a decrease in glomerular filtration rate (gfr), may suggest ph . Di lullo et al . Presented in 2011 the results of tapse in hemodialysis patients . The study included 20 hemodialysis patients aged 5110 years with mean time on dialysis 248 months, without prior medical history of cardiovascular diseases . The authors observed a significantly lower value of tapse (<15 mm) and larger dimensions of the right heart chambers in patients with proximal avf compared with a group of 10 patients with hemodialysis access consisting of permanent catheter inserted through the internal jugular vein to the superior vena cava . According to doppler echocardiography in the population of patients with proximal avf single session of hemodialysis did not affect the pressure in the pulmonary artery, but was associated with a decrease in the value of tapse (163 mm). Suggest a significant correlation between the type of avf and pathogenesis of ph in hemodialysis patients . Similar conclusions emerge from the study by beigi et al . In the group of 34 hemodialysis patients high flow avf formed on the brachial artery (30 patients; mean blood flow1463 ml / min) showed a significantly higher pulmonary artery pressure in the doppler assessment in relation to the low flow avf formed on the radial artery (4, patients; mean blood flow 422 ml / min). The available literature does not provide much data on the prevalence of ph in a population of patients with esrd treated with peritoneal dialysis . Bozabas et al ., in 2009, analyzed a population of 500 dialyzed patients including 432 on hemodialysis and 68 on peritoneal dialysis . Echocardiographic diagnosis of ph was made in 17% (n=85) of all examined patients . Pulmonary hypertension was more frequently diagnosed among hemodialysis patients (19%, n=81) than in patients treated with peritoneal dialysis (6%, n=4). Their 2012 study included 66 dialyzed patients (mean age 57 years, 34 patients treated with hemodialysis, 32 with peritoneal dialysis). The mean duration of treatment on dialysis for both groups was respectively 102 and 44 weeks . Echocardiographic features of ph defined as pasp 35 mmhg (mean pasp was 37.5 mmhg) were found in 30% of a group (n=20). Ph occurred significantly more often in patients on hemodialysis (41%, n=14) compared to patients treated with peritoneal dialysis (19%, n=6) (p=0.04). Among hemodialysis patients diagnosed with measurement of tapse represents a novelty in the diagnosis and monitoring of ph in dialyzed patients . Implementation of this technique into everyday practice does not require sophisticated equipment or prolonged image analysis and is fairly simple even for less experienced physicians . Another advantage is the fact that tapse can be determined in patients with atrial fibrillation with a high ventricular rate in whom reliable echocardiographic examination, including doppler measurements, may be difficult to perform . Tapse should be measured in the apical four - chamber projection using a one - dimensional echocardiography (m - mode). Tapse value determines the range of motion or displacement of the tricuspid valve ring in the longitudinal plane in successive phases of the cardiac cycle, reflecting the function of the right ventricle . Forfia et al . In a prospective study of 63 patients diagnosed with ph showed that the value of tapse less than 18 mm was an independent risk factor for mortality in this population . Table 3 summarizes the normal values of basic echocardiographic measurements for assessing the structure and function of the right heart . In a group of 202 patients with stage 3 ckd and a negative history of ph discovered reduced values of tapse (<23 mm) in 43% of the studied group . Tapse showed a statistically significant negative correlation with the values of systolic blood pressure in the pulmonary artery . Consequently, statistical analysis enabled isolation of subpopulation of patients at risk of developing pulmonary hypertension . This group was characterized by low values of tapse (around 16 mm) and high values of systolic blood pressure in the pulmonary artery (about 38 mmhg). It should be pointed out, that the study by floccari et al . Included patients in the early stages of ckd, which means not on dialysis . The strength of this study was the identification a subpopulation of patients at risk of developing ph . According to the authors these patients in the case of sudden progression of ckd and need for dialysis should not be qualified to proximal, i.e., high avf but either for a distal avf, or for peritoneal dialysis . The authors noted also that in the case of congestive heart failure, shortness of breath and a decrease in daily diuresis, aside from an effect of a decrease in glomerular filtration rate (gfr), may suggest ph . Di lullo et al . Presented in 2011 the results of tapse in hemodialysis patients . The study included 20 hemodialysis patients aged 5110 years with mean time on dialysis 248 months, without prior medical history of cardiovascular diseases . The authors observed a significantly lower value of tapse (<15 mm) and larger dimensions of the right heart chambers in patients with proximal avf compared with a group of 10 patients with hemodialysis access consisting of permanent catheter inserted through the internal jugular vein to the superior vena cava . According to doppler echocardiography in the population of patients with proximal avf single session of hemodialysis did not affect the pressure in the pulmonary artery, but was associated with a decrease in the value of tapse (163 mm). Suggest a significant correlation between the type of avf and pathogenesis of ph in hemodialysis patients . Similar conclusions emerge from the study by beigi et al . In the group of 34 hemodialysis patients high flow avf formed on the brachial artery (30 patients; mean blood flow1463 ml / min) showed a significantly higher pulmonary artery pressure in the doppler assessment in relation to the low flow avf formed on the radial artery (4, patients; mean blood flow 422 ml / min). The available literature does not provide much data on the prevalence of ph in a population of patients with esrd treated with peritoneal dialysis . Bozabas et al ., in 2009, analyzed a population of 500 dialyzed patients including 432 on hemodialysis and 68 on peritoneal dialysis . Echocardiographic diagnosis of ph was made in 17% (n=85) of all examined patients . Pulmonary hypertension was more frequently diagnosed among hemodialysis patients (19%, n=81) than in patients treated with peritoneal dialysis (6%, n=4). Their 2012 study included 66 dialyzed patients (mean age 57 years, 34 patients treated with hemodialysis, 32 with peritoneal dialysis). The mean duration of treatment on dialysis for both groups was respectively 102 and 44 weeks . Echocardiographic features of ph defined as pasp 35 mmhg (mean pasp was 37.5 mmhg) were found in 30% of a group (n=20). Ph occurred significantly more often in patients on hemodialysis (41%, n=14) compared to patients treated with peritoneal dialysis (19%, n=6) (p=0.04). Among hemodialysis patients diagnosed with the achievements of contemporary echocardiography, especially the introduction of measurement of tapse in the early diagnosis of pulmonary hypertension in a population of patients with chronic kidney disease, particularly esrd requiring dialysis, showed a significant diagnostic and prognostic value of this simple test . In patients with esrd at risk of developing pulmonary hypertension if ph was diagnosed in a patient treated with hd, it facilitates a decision to change the treatment modality to peritoneal dialysis or qualification for earlier kidney transplantation.
Chronic obstructive pulmonary disease (copd) is presented mainly by bronchitis and emphysema in various combinations, often with asthmatic complications . Development of cor pulmonale and/or upper respiratory infections may exacerbate the clinical outcome in chronic course of the disease . Clinical examination as well as objective quantification of oxygenation is often critical for the clinical management of the patients . Arterial blood gas sample is often painful and carries the risk of local hematoma, infection and occlusion / embolization with consequent ischemic injury . Hand - held pulse oximeters are usually applied in primary care due to small size, user - friendliness and affordable prices . A meta - analysis of published studies has found a weighted mean correlation coefficient of 0.895 between the pulse oximetry and arterial blood sample . In addition, pulse oximetry can be used in the patients with acute respiratory insufficiency and in the follow - up of chronic respiratory conditions or acute exacerbations of copd . The available literature suggests that the use of pulse oximetry for copd patients would be limited to acute events or specific situations . The study was aimed to explore the association between spo2 and spirometer parameters of disease severity in copd patients with a view to identify whether the pulse oximetry can be used as an alternative to arterial values in the clinical management of copd patients in routine practice . The study sample comprised of 31 patients diagnosed with copd, according to the criteria established by the global initiative for chronic obstructive disease (gold) who were consecutively referred to the respiratory outpatient clinic of a university hospital between may 2011 and december 2012 . All the patients were subjected to full medical history, general and local chest examination and chest x - ray . Spo2 saturation was acquired for every patient using a portable pulse - oximetry device (oxipen, envitecwismar, germany). The percentage of hemoglobin oxygen saturation was measured after connecting the optical diodes on the patients fingers . Each experiment was performed 3 times and the mean value was considered as the spo2 . In the next step, data regarding the spirometry [forced expiratory volume in 1 second (fev1) and forced vital capacity] in all the patients were measured . Based on the gold criteria, copd was considered very severe (gold iv) if fevi / fvc <0.7 and fev1 <30% predicted or fev1 <50% predicted with respiratory failure or signs of right heart failure; severe (gold iii) if 30 fev 50%; moderate (gold ii) if 50 fev 80%, and mild (gold i) if the fev 1 /fvc ratio was <70 and fev 1 80% of the predicted value (8). Otherwise they were considered to have no lung disease (normal) or gold stage 0 if they reported respiratory symptoms . The study complies with the declaration of helsinki and was approved by the institutional ethics committee of our university, and all patients gave written informed consent . For the statistical analysis, the shapiro - wilk test was used to determine the normality of the quantitative variables . The data were expressed as median, interquartile range; man - whitney u test and kruskal - wallis test were applied for quantitative comparison, and differences in categorical variables were examined using chi - square test . Statistical analysis was performed using an ibm computer and pasw software, version 18.0 (spss, inc ., the study sample comprised of 31 patients diagnosed with copd, according to the criteria established by the global initiative for chronic obstructive disease (gold) who were consecutively referred to the respiratory outpatient clinic of a university hospital between may 2011 and december 2012 . All the patients were subjected to full medical history, general and local chest examination and chest x - ray . Spo2 saturation was acquired for every patient using a portable pulse - oximetry device (oxipen, envitecwismar, germany). The percentage of hemoglobin oxygen saturation was measured after connecting the optical diodes on the patients fingers . Each experiment was performed 3 times and the mean value was considered as the spo2 . In the next step, data regarding the spirometry [forced expiratory volume in 1 second (fev1) and forced vital capacity] in all the patients were measured . Based on the gold criteria, copd was considered very severe (gold iv) if fevi / fvc <0.7 and fev1 <30% predicted or fev1 <50% predicted with respiratory failure or signs of right heart failure; severe (gold iii) if 30 fev 50%; moderate (gold ii) if 50 fev 80%, and mild (gold i) if the fev 1 /fvc ratio was <70 and fev 1 80% of the predicted value (8). Otherwise they were considered to have no lung disease (normal) or gold stage 0 if they reported respiratory symptoms . The study complies with the declaration of helsinki and was approved by the institutional ethics committee of our university, and all patients gave written informed consent . For the statistical analysis, the shapiro - wilk test was used to determine the normality of the quantitative variables . The data were expressed as median, interquartile range; man - whitney u test and kruskal - wallis test were applied for quantitative comparison, and differences in categorical variables were examined using chi - square test . Statistical analysis was performed using an ibm computer and pasw software, version 18.0 (spss, inc ., the study was conducted on 31 patients with copd, including 24 males and 7 females (mean age: 55.09 11.61; range: 35 - 76 years). The pulse oximetry and spirometric variables of the patients are described in the table 1 . Physiological variables of the included patients there was not a significant association between fev1% predicted and spo2% (p value> 0.05), while there was a significant association between fev1/fvc% predicted and spo2% (r = 0.556, p value <0.001) [figures 1 and 2]. The dotted curves represent the 95% confidence intervals of fev1% for a given spo2% correlation between fev1/fvc% predicted and spo2% in the included patients . The dotted curves represent the 95% confidence intervals of fev1/fvc% for a given spo2% in addition, the association among age with spo2%, fev1% predicted and also fev1/fvc% predicted was not significant statistically (p value> 0.05). The median fev1% predicted in the stage 0 was [median 90%, interquartile range (90%-95%)]; stage i was 83%, 75%-89%; stage ii was 62%, 51%-79%; stage iii was 45%, 38%-48% and also stage iv was 27%, 21%-33%, which was a significant differences among them in five stages of gold using kruskal - wallis test (p value <0.05). The median fev1/fvc% predicted in the stage 0 was [median 74%, interquartile range (73%-89%]; stage i was 72.5%, 69%-78%; stage ii was 70%, 58%-79%; stage iii was 56%, 52%-70% and also stage iv was 57.5%, 47%-68%, which was a significant differences among them in five stages of gold (p value <0.05). The median spo2% in the stage 0 was [median 94%, interquartile range (93%-97%)]; stage i was 94.5%, 91%-99%; stage ii was 92%, 86%-96%; stage iii was 90%, 74%-96% and also stage iv was 93.5%, 93%-94%, which was not a significant differences among them in five stages of gold (p value> 0.05). Copd is the sixth prominent cause of death in the world, and is estimated to be the third killing and the fifth debilitating disease by the year 2020 . Direct measurement of the pulmonary function (spirometry) and arterial blood gas (abg) analysis are indicated for the clinical management of copd . Although pulse oximeters are extensively used in emergency departments, anesthesiology, and critical care, data on their efficacy in general practice is limited . In this regard, studies have shown that these devices are only reliable when saturation is <80% . We aimed to investigate the effectiveness of pulse oximetery for the determination of severity of copd and respiratory failure . The study showed no correlations between the disease severity (in terms of baseline airflow obstruction) and spo2 in copd patients . The recruited patients in the current study mostly have a stable condition and came to the clinic for their routine follow - up . This finding is in line with a number of studies that have documented a low degree of accuracy for the noninvasive assessment of saturation in stable copd patients . Studied the application of pulse oximetry in 88 patients with deteriorating copd which spearman correlation coefficient for the association between baseline fev1% predicted as documented in the patient's medical file and spo2 was r = 0.55 (p = 0.002) for the patients with acute exacerbations . On the other hand, in 207 patients with stable copd, a weak correlation existed between post - bronchodilator fev1% predicted and spo2 values (spearman r = 0.19, p = 0.006; for current smokers: r = 0.13, p = 0.252 and for former smokers; r = 0.19, p = 0.047). Also, median spo2 values did not differ between gold stages (median test: p = 0.079). These observations suggest that pulse oximetry is mainly helpful when the symptoms are deteriorated because of severe airflow obstruction (fev1% predicted <50%). This is consistent with the findings of previous studies indicating that the rate of tests with spo2 <92% increases when the fev1% predicted value is <50% in stable copd patients . It is noteworthy that our population was consisting of those suffering from mild to severe copd who came to an outpatient clinic . Therefore, the findings of this study may be different from those acquired in hospital - based studies . In this regard, a turkish study in the patients with severe copd demonstrated a high sensitivity for both spo2 and fev1 in reflecting hypoxemia and hypercapnia (83.9 and 90.3%, respectively). Although monitoring of saturation is recommended only for patients with severe disease, pulse oximetry can identify those who might benefit from oxygen therapy or pulmonary rehabilitation . Meanwhile, overestimation of arterial oxyhemoglobin saturation might occur in spo2 values <80%, especially in the patients with darkly pigmented skin . Oxygen saturations are not helpful for the diagnosis of copd; saturations of <98% provides a sensitivity of 79%, but specificity of only 37% . However, pulse oximetry may help in determining the criteria for long - term oxygen therapy or the need for referral to hospital in acute exacerbations . According to dutch family practice guidelines, if pulse oximetry shows spo2 moreover, initiation of oxygen therapy based on an oxygen saturation value <90% is considered another possible application for pulse oximetry in the primary care . Although the current oximeters are practically accurate, they may produce false data in the presence of carboxyhemoglobinemia, hemodynamic instability, dark skin pigmentation, jaundice, and also during the exercise . The oximeter - indicated saturation may significantly overestimate arterial po2, if the patient is alkalemic . Furthermore, the study depicted a significant association between fev1/fvc% predicted and spo2% (r = 0.556, p value = 0.001). The fev1/fvc ratio, also called tiffeneau - pinelli index, is a calculated ratio for the diagnosis of obstructive or restrictive lung disease . It represents the proportion of a person's vital capacity that is exhaled in the first second of expiration . The above - mentioned finding was observed in nomori et al . On 83 patients who underwent lobectomy for lung cancer . A number of studies have showed that fev1/fvc depends significantly on age in healthy subjects . However, the current study did not show the correlation in the copd patients, which could be due to small number of the included cases in our study . Further a larger study may clarify such association in copd patients . In this study the mean age was 55.09 11.61 years with no significant relationship between age and fev1% of predicted in copd patients, and this was matching with the previous studies . Furthermore, the current study may demonstrate that fev1 is essential for the diagnosis and quantification of the respiratory impairment resulting from copd . But, fev1 is known to poorly correlate with symptoms, quality of life exacerbation frequency and exercise intolerance . In total, the study did not support that oxygen saturation measured by pulse oximetry to replace analysis of an arterial blood gas sample in the clinical evaluation of oxygenation in health care patients with copd . Unfortunately, we did not include a standardized assessment of dyspnoea in our study, which would have enabled us to investigate the relationship between the severity of dyspnoea and spo2 . Another limitation is that we did not measure arterial blood gas components, such as pao2, which would have allowed us to acquire more comprehensive results . However, these findings support the claim that spirometric parameters, but not spo2 level, could be useful indicators of the patient outcome, although further evidence needs to be obtained . The study may demonstrate that oxygen saturation measured by pulse oximetry appears to be independent of the degree of airways obstruction as quantified by the fev1; although further evidence needs to be assessed these preliminary findings.
The feasibility and effectiveness of vision screening programs for detection of glaucoma, diabetic retinopathy (dr), cataracts, age - related macular degeneration (amd) have been described [13]. The majority of such programs employ digital color fundus imaging to determine whether a follow - up examination with an ophthalmologist or optometrist is required . Several investigators initiated studies examining the use of additional technologies and imaging modalities, including software - assisted digital filters, retinal thickness analyzer, and slit lamp microscopy to enhance the quality of obtained images aimed at improving detection and characterization of vision - threatening diseases (vtds) [46]. In addition, using a structured questionnaire to assess patient satisfaction attending a teleophthalmology versus conventional (i.e., in - person) screening for dr, kumari rani et al . Found that 34% of participants preferred teleophthalmology to conventional screening and 61% felt that both types of screenings were equally satisfying . In this and other studies, the most common reasons given for improved patient satisfaction with teleophthalmology versus conventional screenings were cost reduction, ability to view images directly, decreased travel time, and direct consultation with a clinician [7, 8]. Fundus autofluorescence (faf) is a specific wavelength light emission from lipofuscin and other molecules that accumulate in retinal pigment epithelial (rpe) cells secondary to photoreceptor oxidative damage . Specific faf patterns have been described for numerous ocular diseases and conditions, and they may also be useful in monitoring disease progression . In the current study, we describe and analyze the incorporation of faf technology in our community - based ocular screening program . Screening participants included homeless and working - poor individuals at soup kitchens and churches in essex county, new jersey . Fifty consecutive individuals were enrolled in this prospective, pilot imaging study in march 2011 . A detailed description of our screening program has been published previously . In brief, the screening team consisted of: (1) senior medical students who completed an intake form (personal and family history of medical and ocular problems, smoking history, etc . ), measured blood pressure, pulse, and oxygen saturation, assessed visual acuity (va; simav, padova, italy) and visual fields (frequency doubling technology; carl zeiss meditec, inc ., dublin, ca); (2) an imaging technician who measured intraocular pressure (iop; canon tx - f full auto non - contact tonometer, tokyo, japan), performed nonmydriatic color and faf imaging (canon cx-1 15.1 megapixel camera with cmos sensor, tokyo, japan); and (3) an onsite medical director who analyzed complete screening data, reviewed color and faf images, and made referrals for follow - up examination as needed . Those participants requiring additional follow - up examinations were referred to the eye clinic at the institute of ophthalmology and visual science at university of medicine and dentistry of new jersey (newark, nj) that accepts uninsured patients . Presenting va was defined as the subject's entering vision with distance correction (i.e., contacts or glasses), if applicable . Snellen va was converted into log mar scale, averaged, and then reconverted back into snellen va . By starting the screening process in a staggered fashion (i.e., at different stations), on average, 11 subjects were screened per hour (not including the time required for reviewing screening data and counseling the participants). Based on personal, social, and/or family history, information packets (available in english or spanish) were handed out and participants were counseled accordingly . Nonmydriatic color and faf images were saved and viewed on a high - resolution, wide - screen fujitsu laptop (kanagawa, japan) using digital imaging and communications in medicine (dicom)-compatible canon eye - q software (canon medical systems, irvine, ca). These were evaluated side - by - side for potential changes in the optic nerve head (i.e., cup - to - disc ratio, asymmetry, neuro - retinal rim color, and nerve fiber layer integrity), blood vessels (i.e., artery - to - vein ratio and nicking, microaneurysm formation, and neovascularization), and macula and posterior pole (i.e., pigmentary changes and drusen formation). Following nonmydriatic color imaging, a second set of nonmydriatic images was taken in the faf mode (530/640 nm exciter / barrier filters). Subjects were allowed to dark - adapt for 2 - 3 minutes . In order to prevent accommodation and pupillary constriction, a single photograph was captured per eye at a 300 watts / second flash intensity . Two to three minutes were required in between in order for the eyes to recover . The resulting 256 monochromatic, grayscale images were then analyzed side - by - side with color images . (i.e., amd, drusen), the emphasis was placed on the macular region, which was assessed for changes in fluorescence . Hypofluorescence signified rpe cell death, while hyperfluorescence suggested active expansion of area of geographic atrophy (ga). Hyperfluorescence can be classified according to specific phenotypic patterns that can be associated with amd progression . For glaucoma, the peripapillary region was of particular interest as hyperfluorescence in this area has been shown to correlate with optic nerve atrophy and/or glaucoma progression . This is different from a myopic crescent, which would appear as a gray band typically spanning the temporal peripapillary region . As for dr, we focused on diabetic macular edema (dme) that may appear as areas of hyperfluorescence . Females represented 52% (n = 26). There were 29 (58%) african americans, seven (14%) caucasians, seven (14%) hispanics, and seven (14%) others . Nine (18%) subjects had diabetes mellitus type 2 for a mean duration of 10.5 years; 13 (26%) had hypertension (based on self - reported history and/or blood pressure measurements at the screening); and 16 (32%) were active smokers . Only 14 (28%) had a dilated fundus examination within the past year . Mean sd iop was 14.8 3.9 mm hg and 14.9 4.4 mm hg in the right and left eye, respectively . Performing color and faf imaging on each eye resulted in reviewing 44 images per hour . We identified nine (18%) individuals with distinct color and/or faf patterns representing dr (n = 3), focal rpe defects (n = 2), dme (n = 1), amd (n = 1), central serous retinopathy (csr; n = 1), and ocular trauma (n = 1) (figure 1). In eight (89%) of these subjects, faf imaging improved detection and/or characterization of pathology versus color imaging . For example, in individuals with dr, faf identified a larger number of dot - blot hemorrhages and microaneurysms, and it was superior at outlining rpe disturbance compared to color imaging (figures 2(a) and 2(b)). While only minor perifoveal rpe hypopigmentation was evident on the color image, we identified two patients with extensive focal rpe defects after faf imaging (figures 2(c) and 2(d)). In the subject with amd, faf imaging provided more detail than color imaging in terms of the extent of rpe disturbance (figures 2(e) and 2(f)). In an individual with chronic csr, although we were able to appreciate a circular lesion nasal to the fovea in the color photograph, the faf image was superior at characterizing the full extent of this lesion (e.g., rpe hyperfluorescence) (figures 2(g) and 2(h)). In the case of ocular trauma, extensive fibrosis was appreciated on color imaging, while faf imaging further delineated an extensive area of rpe degeneration (figures 2(i) and 2(j)). The only pathologic finding that was better appreciated on color imaging was dme with extensive hard exudates that were not apparent on faf imaging (figures 2(k) and 2(l)). Based on these findings, seven (14%) subjects were referred to a retinal specialist and two (4%) to a primary ophthalmologist . All subjects were successfully imaged under the study conditions and none were referred for a dilated examination due to small pupil size . As far as we can discern, this is the first prospective pilot imaging study set out to determine the feasibility of integrating faf technology in a community - based ocular screening program . We postulated that correlation of color and faf imaging may allow for improved characterization of damage to the retina and rpe . Tool in several retinal diseases / conditions, including amd, hydroxychloroquine toxicity, and serpiginous - like choroiditis [14, 15]. Logistically during our screening, the process was aided by the fact that the canon cx-1 camera comes specifically configured to perform color, faf, and other types of imaging . Therefore, we did not have to be concerned about transporting an extra piece of equipment to our screenings (which commonly occur in difficult to access locations such as basements or buildings without elevators), or decreasing the number of participants screened due to space restrictions . Additionally, the inter - phased imaging software allowed for side - by - side viewing of color and faf fundus images, which aided in comparing and correlating the ocular findings . An average of 22 eyes were imaged, and 44 color and faf digital images were reviewed per hour . Identification of vtds necessitating referral to a general or specialty ophthalmologist occurred in nine (18%) individuals . In our previous studies, an average of 2630 eyes was imaged per hour and the rate of vtd detection was 1030%; however, neither of these studies employed two different types of imaging modalities [11, 16]. Of the nine (18%) individuals with identified vtds in our current study, only seven (14%) would have been referred for a comprehensive examination based on results of color fundus imaging (excluding the two participants with focal rpe defects). Thus, in addition to improved characterization of pathology identified on color fundus imaging, incorporation of faf imaging as a complementary technology in this cohort increased ocular pathology detection in our screenings by 29% (from seven to nine eyes with pathology). In the present study, we were able to show successful integration of faf imaging in a community - based ocular screening program . The imaging itself was simple to perform and it resulted in enhanced detection of abnormalities in a wide variety of retinal diseases when performed side - by - side with color fundus imaging . Some of the potential limitations to using faf technology in screenings include: (a) increased cost of the faf camera versus a color fundus camera; (b) increased screening time to allow for reversal of pupil constriction secondary to higher flash intensity required to acquire faf images; (c) our currently limited understanding of the utility of faf in certain vtds like glaucoma and dr; (d) requirement of patient cooperation and clear ocular media (e.g., without a visually significant cataract or vitreous opacity) in order to obtain high - quality images; and (e) potential difficulty with image interpretation due to absence of standardized equipment, protocols, and grading systems . A cost - benefit analysis, including the final impact on local health indicators, should be pursued . Although the faf capable camera used in the present study is more expensive than a conventional color fundus camera, the potential of faf to improve ocular disease detection warrants further study . This may be especially pertinent for retina specialists for the diagnosis, detection of progression, anticipation of prognosis, and treatment of amd, dme, and so forth . Earlier detection may positively impact the exorbitant costs attributable to undiagnosed ocular diseases such as amd, glaucoma, and dr [1720]. Future work correlating faf findings with optical coherence tomography may enhance evaluation of diseases that damage the retina, rpe, and choroid, thereby potentially avoiding false positive referrals . Based on our findings, we believe that faf imaging is a complementary technology that may have a significant impact on teleophthalmology applications . Advances in imaging technologies make screening for vtds more accurate by employing complementary imaging technologies such as color fundus imaging, software - assisted analysis, and faf . In the current study, the number of subjects allowed for a descriptive analysis only . A larger follow - up study designed to compare the sensitivity, specificity, and positive and negative predictive values for vtd detection with and without the incorporation of faf is warranted . More specifically, a prospective, double - armed sequential screening study in which referral patterns based on color fundus imaging only versus color fundus imaging followed by faf imaging should be performed . The potential impact of improved characterization and detection of ocular pathology on disease burden (including cost to society) warrants further investigation . As a corollary, we believe that it would be pertinent to conduct cost analysis studies in order to make recommendations regarding the economic feasibility of incorporating faf imaging in ocular screening programs.
A vasoactive intestinal polypeptide (vip)-producing neuroendocrine tumour (vipoma) is an extremely rare tumour that presents with symptoms such as watery diarrhoea and hypokalaemia as a result of vip overproduction . Surgery is generally the preferred treatment, but molecular - targeting drugs such as everolimus and sunitinib have been recently developed for cases of advanced disease . Furthermore, for cases where radical resection is impossible, multimodal therapy including surgical reduction of the tumour mass is now recommended (1). We treated a case in which good disease control was achieved through transcatheter arterial embolization (tae) of liver metastases in a patient with a pancreatic vipoma who had developed somatostatin resistance . We report the course of this patient's treatment, which may be useful when selecting a therapy for vipoma that is difficult to treat surgically . He presented at his local doctor's office with a chief complaint of watery diarrhoea occurring 10 times per day, which had persisted for approximately 6 months, in combination with epigastric discomfort . Iu / l), a serum potassium concentration of 2.4 mmol (range: 3.5 - 5.0 mmol), and a urea nitrogen level of 21 mg / dl (range: 6 - 20 mg / dl) (table 1). Computed tomography (ct) showed multiple tumours in both lobes of the liver (fig . A-2: a tumour 30 mm in diameter could be seen in the tail of the pancreas . B-1: computed tomography revealed decreased sizes of all of the tumours from 2010 after the administration of octreotide . B-2: marked reduction was also seen in the size of the tumour in the pancreatic tail . C-1: a slight increase in the size of the liver tumours was seen on computed tomography from 2014, when symptoms recurred . C-2: the tumour in the pancreas also increased in size, which was comparable to that of the first image obtained 4 years previously . D-1: after the administration of everolimus, the metastatic liver tumour shrank in size . Contrast - enhanced abdominal ct performed at our hospital revealed a hypervascular hepatic mass that stained in the early arterial phase in both lobes . Although the hepatic mass was suspected to be a metastatic tumour, the primary tumour was not identified at this point, with the mass identified at the tail of the pancreas considered to be a swollen lymph node . Fluorodeoxyglucose positron emission tomography (fdg - pet) did not clearly demonstrate a primary tumour . The ki-67 index was 5% positive, and immunostaining was positive for synaptophysin and chromogranin . Serum vasoactive intestinal peptide (vip) was high at 553 pg / ml (normal 100 pg / ml). Because the mass was diagnosed as a functional neuroendocrine tumour (net; vipoma) with an unknown primary, treatment with an analogue of somatostatin (subcutaneous injection of sandostatin 50 g, twice daily) was initiated in june 2010 . The mass decreased in size, and the clinical symptoms improved . Thereafter, maintenance therapy with a long - acting form of octreotide (sandostatin lar, 30 mg intramuscularly once monthly) was prescribed, which sustained the improvement in symptoms and tumour size on ct (fig . 1b-1, 1b-2). Although we considered surgical treatment, the patient did not consent to this procedure, and pharmacological treatment was thus continued . However, in may 2014, 4 years after the initial treatment, the patient once again began to experience watery diarrhoea 10 times per day . A ct examination at that time revealed the enlargement of multiple hypervascular tumours in both lobes of the liver and a tumour in the pancreatic tail (fig . In july of the same year, endoscopic ultrasound (eus) demonstrated an oval, 28-mm - diameter, solid mass with a clear boundary in the tail of the pancreas . Endoscopic ultrasonography - guided fine - needle aspiration (eus - fna) of the mass showed atypical cells with naked nuclei and eccentric nuclei on hematoxylin and eosin (h&e) staining . The ki-67 index was 10% positive, and immunostaining was positive for synaptophysin, chromogranin, as well as vip . A diagnosis of pancreatic net (p - net; vipoma) was made, with a world health organization (who) classification of g2 (fig . 2, 3). A hypoechoic tumour 20 mm in diameter with distinct borders was seen in the pancreatic tail on endoscopic ultrasound . Histopathologic features of the pancreatic neuroendocrine tumour . A: 40, approximately 10% of the tumour cells appeared mib-1 positive on hematoxylin and eosin staining, so the tumour was classified as g2 . B: 40, on vip immunostaining, the cytoplasm was stained granularly and diffusely . The patient was thought to have developed resistance to octreotide and was started on everolimus (afinitor at 10 mg daily [2 capsules per dose]) in july 2014 . After the administration of everolimus, the metastatic liver tumour decreased in size . However, there was no notable improvement in the watery diarrhoea or hypokalaemia . To relieve his endocrine symptoms, tae of the hepatic artery abdominal angiography in september 2014 revealed at least three deeply stained tumours in the right hepatic artery region and multiple small tumour stains around the largest tumour in s6 of the liver (fig . The diarrheal symptoms disappeared the following day, and the serum vip concentration markedly dropped to 83 pg / ml . Four months later, the symptoms recurred, and tae was repeated to treat the remaining liver metastases . The patient continues to take oral everolimus, his symptoms have subsided, and the reduction in tumour size is being maintained . Switching treatment to sunitinib was considered . However, because the liver tumour mass had decreased in size, we opted for continuation of everolimus treatment and management of the endocrine symptoms with tae (fig . Hypervascular tumour staining was seen in three places in the right hepatic artery region . The incidence is extremely low at just 1 in 10 million people per year (3). According to a 2010 survey by ito et al . In japan, vipomas accounted for 0.6% of the p - nets overall, with distant metastases present at the time of diagnosis in 80% of cases (4). The vip produced by these tumours causes the characteristic watery diarrhoea, hypokalaemia, and achlorhydria syndrome (5). The disease is diagnosed on the basis of these symptoms, an elevated serum vip concentration, and tissue immunostaining . In recent years, eus - fna has been increasingly used, as it allows for a histopathological diagnosis but is less invasive than conventional open or laparoscopic biopsy . In terms of fna histological diagnosis, results have been favourable, with a reported sensitivity of 82.6 - 100% and a diagnostic accuracy of approximately 83.3 - 97.3% . Fna offers a high diagnostic performance for detecting p - nets (6 - 11). However, because reports have indicated that lesions in the head of the pancreas and tumour masses rich in fibrosis are difficult to diagnose using fna (10), a multiple - modality approach is required for diagnosing such tumour masses . Furthermore, the grade classification of p - nets is determined by the mitotic rate and ki-67 index on a pathological examination . Because of the unevenness of the ki-67 index within the tumour masses, it is preferable to base the grading of net on 2000 cells from an fna sample (11). Other tests for nets include somatostatin receptor scintigraphy and tetraazacyclododecane - tetraacetic acid - modified tyr - octreotide - pet, which have high positivity rates for detecting highly differentiated nets . Fdg - pet is very sensitive for undifferentiated neuroendocrine carcinomas with a high proliferative capacity (2010 who net classification). Functional nets are treated surgically when there are no liver or distant metastases . In cases accompanied by liver and/or distant metastasis, for which a radical cure cannot be anticipated, surgery to reduce the size of the original tumour by at least 90% appears to be effective from the viewpoint of alleviating symptoms (12). Furthermore, for p - nets with distant metastases, it has been reported that resection of the primary tumour is effective for prolonging survival . However, because there are few clinical studies comparing cases with and without primary tumour resection, this issue remains unresolved . A systematic review concluded that resection of the primary tumour for patients with p - nets and unresectable liver metastases should be considered only in high - volume referral centres with strict selection criteria and in a multidisciplinary setting (13). Thus, resection of the primary tumour should be considered for functional p - nets with multiple liver metastases, such as in the present case . However, because our patient refused surgery, we selected medication - based treatment instead . With unresectable functional nets, somatostatin analogues are useful for treating the symptoms (14 - 16) however, resistance may develop as a result of the down - regulation of somatostatin receptors following the long - term use of the drug (17,18). In the present case, the patient developed resistance to octreotide over the course of four years, thus leading to symptom recurrence . The molecular - targeting drugs everolimus and sunitinib have recently been found to be effective antitumour drugs against primitive nets (g1/g2) (19,20). A phase iii comparative trial (radiant-3) demonstrated the effectiveness of everolimus, with treated subjects showing a progression - free survival of 11 months, compared with 4.6 months in those taking the placebo (19). Sunitinib was similarly found to offer better progression - free survival than a placebo (11.4 vs. 5.5 months) (20). Tae and transcatheter arterial chemoembolization (tace) have also been found to be effective as local treatments for net liver metastases (7). Tae in particular may be about 90% effective in providing complete or partial relief of endocrine symptoms, an effect reported to last 6 - 27 months (21). Tae led to an improvement in our patient's symptoms as well; however, symptom recurrence after four months necessitated repeat tae . Tae may thus be an effective treatment for liver metastases, although the results may be only temporary, and instances of recurrence may require treatment be conducted repeatedly . During the 30-year period from 1985 to 2015, we identified 6 cases of vipoma in the english literature (based on a pubmed search) that were managed by non - surgical multidisciplinary modalities alone (22 - 26). All patients had functional net with watery diarrhoea and were found to have liver metastases . Survival for at least 10 years was achieved in 3 cases with drugs or drugs plus tace . Two patients died: one with severe hypokalaemia associated with watery diarrhoea (22) and one whose increasingly severe diarrhoea could no longer be controlled, despite the use of a somatostatin analogue and repeated tace (24). The fact that exacerbation of symptoms was the cause of death in two cases and that survival of at least 10 years was seen in other cases as a result of a combination of chemotherapy and tace suggests that control of endocrine symptoms in unresectable vipoma is important to improve the prognosis . In the present case, the endocrine symptoms are currently being controlled with oral everolimus, after tae twice for multiple liver metastases; the patient appears to have a good long - term survival . Furthermore, as the main lesions responsible for the endocrine symptoms in our patient are likely the liver metastases, if symptoms recur in the future, we expect that we will be able to control the disease with aggressive tae . The successful control of endocrine symptoms with sunitinib has been reported in similar cases (27). Thus, if combination treatment with everolimus and tae is problematic, switching of the drug regimen may be necessary . Unresectable vipoma reported between1985 and 2015 . In conclusion, surgery is certainly the best current treatment for vipoma if it can be completely resected . However, in unresectable cases, non - surgical multidisciplinary treatment that controls endocrine symptoms may result in a long - term survival.
Gout is a common inflammatory arthritis that is characterized by the deposition of monosodium urate crystals in the synovium . An estimated 6.1 million adults in the united states have gout.1 monosodium urate crystals have decreased solubility at lower temperatures, and consequently, the most commonly affected sites are peripheral joints . Pseudogout is the deposition of calcium pyrophosphate dihydrate crystals in the joints or periarticular structures, which leads to inflammation in joints . Occurrence of gout in the spine is rare, with little over 70 cases reported in the literature . One review of the literature found that the lumbar spine was most commonly affected (56% of cases), followed by the cervical and thoracic spine, which both occurred in 22% of cases.2 out of these cases, the most common presenting symptoms were nonspecific paraparesis (39.0%), radiculopathy (27.0%), and back pain (18.0%).3 given the rare incidence and nonspecific presentation, an inclusion of gout in the differential diagnosis of atypical neurocompressive pathologies is necessary for diagnosis . In the spine, gout may affect the epidural space, ligamentum flavum, intervertebral disc, pedicles, facet joint capsule, and neural foramen.4 this may present as spinal stenosis, lumbar radiculopathy, spondylolisthesis, or cauda equina syndrome . Histologic findings show a granulomatous infiltrate of multinucleated giant cells, histiocytes, and fibroblasts . In addition, specimens examined under polarized light microscopy demonstrate the characteristic needle - shaped crystals with negative birefringence of gout . High serum urate levels, a concomitant history of advanced renal disease, previous gout attacks (podagra), and the cutaneous manifestation of tophi all point to the diagnosis . A 24-year - old man with a 4-year history of tophaceous gout and chronic kidney disease presented with a 3-year history of lower back pain that acutely worsened over the course of a week before hospital admission . He had developed shooting pain down the lateral aspect of his thigh with the right worse than the left . Of note, the patient had not been taking any of his gout medication for the six months before admission . He has had several acute flares of gout since his diagnosis and has disabling tophi in his hands . Physical exam was notable for weakness of the right lower extremity, with 4/5 strength in ankle dorsiflexion, extension of the big toe, and ankle plantarflexion . His creatinine on admission was 4.80 mg / dl and serum uric acid was 14.6 mg / dl . 1), demonstrating a large, heterogeneous, infiltrating calcified soft tissue mass in the lumbosacral region, expanding the neural foramen, and scalloping the vertebral body, with erosion of the facet joints . Magnetic resonance imaging (mri) revealed an isointense mass surrounding the l5 nerve root on t1-weighted images (fig . Tissue diagnosis was then indicated although the concordance of imaging with the patient's history and physical exam suggested gout . The patient underwent ultrasound - guided fluid aspiration, which revealed negatively birefringent crystals on polarized light microscopy, confirming the diagnosis of gout (fig . 4). Axial noncontrast ct at the l5-s1 level shows a hyperattenuating, partially calcified soft tissue mass scalloping the posterior vertebral body, widening the right neural foramen, and eroding the right facet joint . Axial t1-weighted mri at the l5-s1 level demonstrates a heterogeneous soft tissue mass mostly isointense to muscle surrounding the exiting right l5 nerve root and extending anteriorly into the retroperitoneum, with an epidural component causing dural compression and displacement . The solid components fill the right l5-s1 and s1 - 2 neural foramina, with scalloping of the vertebral bodies . Polarized light microscopy of ultrasound - guided fluid aspirate of paraspinal mass reveals numerous needle - like negatively birefringent monosodium urate crystals . Intraoperatively, there was significant marginal sclerosis in areas of the sacrum, especially the right sacral ala and s1 foramen . The mass had also grown into the paraspinal muscles and into the retroperitoneum along the l5 nerve root . A decompressive laminectomy of l4-s1 and resection of intraspinal canal lesion and perineural lesion was performed (fig . Large aggregates of chalky tophi and a cystic collection of milky white fluid were intralesionally resected . Postoperatively, the patient was restarted on colchicine and allopurinol and given a brief burst of prednisone . By the 6-week clinic visit, he had regained his strength in his right lower extremity and had interval resolution of his lower back pain . Surgical pathology specimen: en bloc resection of a nodular, chalky soft tissue mass . As mentioned, gout rarely affects the spine and even more rarely evolves in such an aggressive fashion . Initially, it was felt that imaging and clinical symptoms favored malignancy given the progressive neurologic deficit and imaging characteristics . Physical exam and plain films revealed the presence of multiple gouty tophi in other small joints, which increased suspicion for gouty arthropathy of the spine (fig ., isolated spinal gout can be extremely difficult to diagnose and as such a tissue diagnosis before intervention is highly encouraged except in the most urgent / emergent scenarios . Oblique radiograph of the right wrist obtained 3 years prior shows classic features of gout, including juxta - articular punched out erosions without osteoporosis at the second metacarpophalangeal joint and large calcific soft tissue tophus at the ulnar side of the wrist . Although crystal deposition disease of the spine is rare, the diagnosis can be made by the patient's history, the results of computed tomography (ct) or mri of the spine, and tissue samples . Gouty tophi normally appear as a hypointense, homogenous mass associated with a joint on t1- and t2-weighted mr images, which enhances with gadolinium because of vascularized reactive tissue in the tophus.5 6 imaging characteristics of spinal gout are nonspecific, however, and the use of an image - guided fine needle aspiration or biopsy is recommended . The imaging characteristics in our case were somewhat unique given the heterogeneity of the mass demonstrating discrete areas of soft tissue, calcification, and cyst formation, raising the initial concern of a nerve sheath tumor with possible malignant degeneration . The mass was noted to extend along the right - sided l5 and s1 nerve roots . Also, we believe this massive spinal tophus to be the largest reported, measuring 7.5 cm 2.8 cm 10.3 cm, and extending from the level of l3 to s1 in the sagittal plane . In most cases, diagnosis can only be confirmed by histological examination of biopsy material . This minimally invasive diagnostic test allows differentiation from other important imitators such as neoplastic disease or infection and avoids unnecessary exploration . Dual - energy ct may have a future role in differentiating spinal gout from infection or tumor and obviating biopsy by identifying the presence of uric acid in tissues . Although some controversy exists, most presentations of axial pain caused by spinal gout can be medically managed.3 patients without concomitant neurological deficits can be treated medically after spinal infection is excluded . Medical treatment with nonsteroidal anti - inflammatory agents, such as naproxen and ibuprofen, and steroids, such as prednisolone, are effective to control pain acutely . This is followed by administration of a urate - reducing agent for long - term control such as probenecid, allopurinol, or rasburicase . Maintaining adequate hydration and alkalization of urine is required to prevent recurrent episodes.3 5 it is important for the clinician to realize that surgery is only warranted for relief of neurologic symptoms and that long - term management hinges on adequate medical therapy . Given the long duration of this patient's axial skeletal pain, aggressive medical management may have rendered surgery unnecessary; however, a cautious approach is warranted when neurological deficits are present . Department of rheumatology, chru (university teaching hospital), and universit de franche - comt, besanon, france spine is not the most characteristic location of gout, and this is described mainly in case reports . Jegapragasan et al report a case of tophaceous gout of the lumbar spine, revealed by low back pain and neurological symptoms in a patient with long - standing peripheral tophaceous gout, elevated uric acid levels, and renal impairment, in a context of treatment discontinuation . Imaging revealed a tophaceous mass surrounding zygapophysial joint, and diagnosis was confirmed by needle aspiration, revealing negatively birefringent crystals . Surgery (decompressive laminectomy and resection of intraspinal and perineural lesion) was required in this case, with favorable outcome . The frequency of spine involvement of gout is not clearly established . In a retrospective study of patients with peripheral gout, changes suggestive of gout (tophi or discovertebral erosion) were present in 14% of the cases.1 in a prospective study of 45 patients with at least 3 years duration of poorly controlled peripheral gout, spinal ct evaluation revealed lesions in 35%,2 and only half of them had back pain . Every segment of the spine may be involved, but as recalled by jegapragasan et al, the lumbar segment is the most frequent location . Different features may be seen: vertebral bone tophi, spondylodiskitis, zygapophysial tophi or destructive arthritis, and epidural mass.3 this explains the variety of clinical symptoms (isolated back pain, neurological symptoms associated) and differential diagnosis (infection, tumoral disease, degenerative disease). In one study,2 no differences in age, gout duration, body mass index, renal function, or presence of back pain or hypertension were found between gouty patients with and without spinal lesions, but peripheric radiographic erosions and diabetes were associated with the presence of spine ct abnormalities . The presence of subcutaneous tophi and history of previous gouty attacks are important anamnestic features that may help diagnosis, but nevertheless, gout may be revealed by spinal involvement.3 awareness may help interpretation of imaging (plain radiography, ct, mri). Dual - energy ct may allow in the future a most accurate imaging diagnosis approach of gout by estimates of tophus attenuation . Needle aspiration is possible for zygapophyseal or discal involvement; otherwise, a guided biopsy is required . However, in all the cases, polarized light microscopy examination is mandatory to establish the diagnosis . Management of these patients may vary according to the clinical presentation . In case of neurological symptoms due to tophaceous mass, surgical decompression evidence of spine location of gout requires medical treatment of gout to avoid subsequent potential neurological complications, since old (colchicine and allopurinol) and new therapeutic possibilities are available for treating inflammatory attacks (anti il-1 strategies) and for urate lowering (febuxostat, rasburicase, and pegloticase).4 5 for a variety of reasons, case reports are located near the bottom of the evidence pyramid . That said, they maintain a firm role in our peer - reviewed literature for the value they can provide us when they highlight rarities and raise our awareness for unusual cases that we as clinicians may be exposed to on any given day . The topic of tophaceous gout affecting the spine is such an unusual case; it fascinates with its variability of presentation and clinical course and, therefore, lacks clear diagnostic and treatment guidelines . This well - documented case and thoughtful commentary impresses upon us the importance of a thorough physical examination of spine patients, including integument and joints outside the axial skeleton . It also underscores the importance of being familiar with differential diagnoses as we look at less than straightforward cases . It stands to reason that an earlier diagnosis in this patient would very reasonably have allowed for nonsurgical medical treatment and helped avoid surgery and subsequent disability.
The contour of the lower face, including the lips and soft - tissue chin, is thought to be closely related to the underlying dentoalveolar structure . Both the upper and the lower lips are known to increase in length during growth and maintain a fairly constant vertical relationship with the edges of the corresponding central incisors after their full eruption.1 during lip closure, a subject with a normal maxillofacial skeletal pattern shows negligible contraction of the perioral musculature, and therefore, changes in the contour of the lip and soft - tissue chin are minimal.2,3 on the other hand, many orthodontic patients display visible tension in their soft - tissue chin during lip closure, which is often considered unesthetic . In particular, patients who have a long anterior vertical dimension or protruded incisors with a large interlabial gap show strain of their soft - tissue chin in an effort to close their lips, because the lower lip is elongated and the mentolabial sulcus moves upward and forward.2,4,5 one goal of orthodontic treatment is to achieve a desirable facial profile by obtaining not only satisfactory dental and skeletal relationships but also esthetic soft - tissue components including the lips and chin . Any dental or skeletal factors found to be contributing to either tension in or deformation of the soft - tissue chin must be considered during diagnosis and treatment planning for better outcomes . The influence of diagnostic factors on pretreatment or posttreatment variables can be evaluated through various analytical methods such as logistic and multivariate regression analyses.6,7 however, these analyses often include complicated predictive equations, which occasionally present difficulties in result interpretation . A classification and regression tree (cart) is a useful analytical method that integrates the data of complex regression analyses and converts them into a simple tree model providing information on diagnostic factors in the order of their impact on the dependent variable with cut - off values.8,9 studies using cart as a mean of analytical method for the evaluation of the soft - tissue chin are scarce . The purpose of this study was to investigate the dentoskeletal factors which may predict soft - tissue chin strain during lip closure . Two hundred and ninety - three women with angle's class i or ii molar relationships and aged 18 - 30 years were recruited from three private orthodontic clinics . The exclusion criteria were as follows: (1) previous orthodontic treatment or orthognathic surgery; (2) multiple missing teeth; (3) presence of prostheses in edentulous areas; (4) anb less than 0; (5) anterior edge - to - edge bite or crossbite; and (6) congenital anomalies such as cleft lip and palate . The records included frontal and lateral facial photographs taken with the lips in the closed and relaxed positions and lateral cephalograms obtained with the teeth in centric occlusion . Visually inspected the lips, mentolabial sulcus, and soft - tissue chin of the subjects in these records and classified the subjects into either the no - strain or the strain group according to the soft - tissue chin tension or deformation during lip closure . Cochran's q test was performed to compare the proportion of subjects classified into the strain group by the three examiners: the differences were found to be significant (p = 0.02). Further, mcnemar's test was used to compare the proportion of subjects classified into the strain group between the examiners . Showed no significant difference (p = 0.892), with their concurrence being 81.5% . However, examiner j.p . Showed significant discordance with both examiner y.k . And examiner y.y . (p = 0.036 and 0.014, respectively). The results of these tests suggested that the examiners showed subjectivity in classifying the women into the no - strain or strain group; to enhance the credibility of this research, the data of only 209 subjects (114 and 95 women from the no - strain and strain groups, respectively) who had been unanimously classified by all the three examiners were used . The lateral cephalogram of each subject was traced on standard 0.003 inch acetate tracing paper with a 0.3 mm mechanical pencil and measured on a scale with 0.5 mm and 0.5 intervals by one orthodontist . The description of cephalometric landmarks and references for the skeletal and dental measurements are shown in table 1 . The reliability of the measurements was assessed via intraclass correlation coefficients (iccs) and the wilcoxon signed - rank test . A t - test was performed to compare the differences in the mean values of the variables between the groups . Three variables that did not pass the normality test were analyzed by the mann - whitney u - test . Multivariate analysis of variance (manova) was conducted to determine the intergroup differences in the variables after age adjustment . The chi - square test was carried out to evaluate the significance of the group distribution according to angle's classification of malocclusion . Moreover, logistic regression analysis was used to determine the possible discriminants for each group . Then, a cart model was constructed to obtain cut - off values for the variables selected as predictors . Tracing and measurement errors were determined by retracing 20 cephalograms acquired through systematic random sampling . The results of the wilcoxon signed - rank test reflected no significant differences in the repeated measurements of all the variables (p> 0.05; table 2). The measurements were considered reliable and measurement errors were thought to be minimal because 26 of the 27 analyzed variables exhibited icc values of 0.95 or higher (p <0.001; table 2). As shown in table 3, the mean values of 22 of the 27 variables were significantly different between the groups . Meanwhile, the age differences between the groups were not significant (p = 0.362). Further, in the manova, the values of wilks' lambda indicated that the intergroup differences in the mean values of the variables after age adjustment were comparable to those before age adjustment (p <0.05). According to angle's classification of malocclusion, 124 and 85 subjects had class i and ii malocclusions, respectively . The strain group included a lower number of subjects with class i malocclusion (48 [38.7%] vs. 76 [61.3%]) and a slightly higher proportion of subjects with class ii malocclusion (47 [55.3%] vs. 38 [44.7%]) than the no - strain group . The chi - square test showed that this trend was significant (p = 0.018). The variables analyzed in the forward stepwise (likelihood ratio) logistic regression analysis included the cephalometric measurements, age, and angle's classification of malocclusion (class i or ii malocclusion). Four variables were chosen as the most prominently related factors for the group classification in the final model (table 4): overbite depth indicator (odi), upper incisor to n - pog plane (u1-npog, mm), overjet, and upper incisor to upper lip (u1-upper lip, mm). A logit model using the four selected variables was derived as follows: where " a, " " b, " " c, " and " d " are the coefficient values of odi, u1-npog, overjet, and u1-upper lip, respectively (table 4). The logit model indicated that the lower the odi value and the higher the u1-npog, overjet, and u1-upper lip values, the greater was the probability of the subject being included in the strain group . By inverting the logit model with exponential function, the following equation was obtained: where " s " is the logit model and " pr(sg) " is the probability of classification into the strain group . The prediction rate at which the group classification by using the four selected variables would concord with the actual classification was 81.8% (table 5). The cart model constructed by using the four selected variables provided the cut - off values of these variables (figure 1). The tree building began at the root (or parent) node (node 0), which included all the subjects in the dataset . The best splitter variable for each node was selected to maximize the average " purity " of the two child nodes . The best splitter variable for node 0 was found to be u1-npog, with a cut - off value of 14.2 mm . Therefore, in node 1, 80% of the subjects showing a u1-npog value less than or equal to 14.2 mm would be categorized into the no - strain group, and in node 2, 79.8% of the subjects showing a u1-npog value greater than 14.2 mm would be categorized into the strain group . Accordingly, u1-npog was considered the primary predictor, with the ability to classify approximately 80% of the total subjects into the strain or no - strain group at the value of 14.2 mm and without the necessity of the other variables . The next splitter variable for both node 1 and node 2 was considered to be overjet, although the number and condition of the subjects in these nodes were different (i.e., node 1 contained 120 subjects with a u1-npog value 14.2 mm and node 2 comprised 89 subjects with a u1-npog value> 14.2 mm). The tree building continued until one of the coupled nodes showed 100% purity in the categorization of the groups, as seen in nodes 12, 14, and 15 . The predictability of the group classification according to the cart model is shown in table 6 . During lip closure, the upper and lower lips cover two - thirds and one - third of the maxillary central incisors, respectively, and the main muscle activity is shown by the mentalis, followed by the lower and upper lips.1 the mentalis is essential in both esthetic and functional aspects because it is responsible for the central motion of the lower lip and determining the position of the chin point.4,10 patients with the dolichofacial type or maxillary protrusion tend to overwork the mentalis and lower lip to compensate for the lip incompetence . This condition leads to tension in the soft tissue at the insertion point of the mentalis and dimpling at the soft - tissue chin point, which is considered functionally and esthetically unfavorable.2 - 4 in this study, frontal and lateral extraoral photographs taken with the lips in their resting and closed positions and lateral cephalograms obtained with the teeth in centric occlusion were examined . To eliminate discrepancies related to gender or age, further, only the subjects who were unanimously categorized into the no - strain or strain group by all the three examiners were included to allow the maximal objectivity . To identify the cephalometric measurements highly related to strain of the soft - tissue chin, a cart is used in various dental and medical studies to evaluate diagnostic or predictive factors for a certain condition or disease.11 - 13 this diagnostic tree is a machine - learning method that allows the construction of a prediction model from the data.8,9 it exhibits independent variables (e.g., the cephalometric variables) in the order of their influence on the dependent variables (e.g., no - strain or strain group) in a stratified manner, with the earlier division or node (higher in the tree) demonstrating a greater effect . Its main advantage is that it defines the cut - off values for dividing the parent node into the two child nodes that can simplify the decision for one way or the other . Another advantage is the graphical presentation of the tree diagram, which effectively aids in the interpretation of complex statistical data . The child nodes can each be recursively divided into two nodes, forming additional child nodes with their own splitters and cut - off values . Of the four discriminants selected in this study, the cart model showed that u1-npog had the highest differentiability at a cut - off value of 14.2 mm . In a previous study, subjects with class ii division 1 malocclusion and lip incompetence had higher perioral muscle activities than those with normal occlusion.14 further, in both maxillary and bimaxillary protrusion cases, a greater amount of muscle tension developed from the lower lip to the chin in the closed lip position, because the amount of lower lip movement increased in relation to the degree of maxillary incisor protrusion.3 these studies are highly comparable to the present study as they evaluated asian male and female patients equivalently or female patients only . Accordingly, maxillary incisor protrusion has a notable effect on the lower lips and can lead to increased strain of the soft - tissue chin . Moreover, the degree of the chin prominence relative to the lip and maxillary incisors should not be overlooked . The chin provides a fundamental base on which the lips rely for support and the balance of the lip position heavily depends on the strength of the chin.15 if the chin is too weak, the relative distance to the maxillary incisors would increase, which might cause the lower lip to protrude in order to reach the maxillary incisors during lip closure and lead to soft - tissue tension in the mentalis area . Therefore, the optimal treatment plan to relieve strain of the soft - tissue chin should include retraction of the maxillary incisors or orthognathic surgery such as bimaxillary anterior subapical osteotomy where a more prominent chin profile is preferred.5,16 the next predictive variable in the cart model was overjet, with cut - off values of 4.8 and 4.2 mm . An increased overjet was associated with decreased activity of the upper lip and increased activity of the lower lip; a positive correlation was noted between the overjet and the activity of the lower lip during lip closure, speech, and mastication.14 otuyemi17 reported that in a preadolescent nigerian population, an overjet greater than 6 mm was strongly related with a " trapped " lower lip and that greater than 7 mm led to inadequate coverage of the maxillary incisors by the lower lip . On the other hand, a direct correlation between the varying overjet and the different upper lip positions has not been shown, confirming a strong association between the overjet and the lower lip rather than with the upper lip.18 although these studies were performed with varying age groups and included male subjects, the results of the present study are in agreement with the previous findings concerning the influence of a large overjet on the movement of the lower lip . Therefore, acquiring a normal overjet may minimize malfunction of the lower lip and distortion of the mentalis . U1-upper lip (mm) is a measurement reflecting the relative length of the lip . Its average values in the strain and no - strain groups were 4.07 and 2.83 mm, respectively, indicating that maxillary incisor extrusion coincides with increased strain in the mentalis . This relationship was significantly different in the subjects with class ii division 1 and class i malocclusions, implying that the maxillary incisors tend to supra - erupt in class ii division 1 malocclusion.2 hayashida et al.19 reported that the most influential factor for the vertical position of the lower lip is the varying position of the tip of the maxillary incisor . An extruded maxillary incisor causes cushioning of the lower lip on the lingual surface and leads to eversion of the lower lip and distortion of the mentolabial sulcus and mentalis . Therefore, the vertical position of the maxillary incisors should also be considered together with the smile arc in the esthetic aspect of orthodontic treatment.2,20,21 the odi was the only skeletal factor among the selected predictors and its influence was not as conspicuous as that of the other variables, which are related to the position of the maxillary incisors . Although its influence is limited, the treatment plan should be oriented toward increasing the odi value to decrease tension in the mentalis . However, this may be difficult to achieve because the ab plane angle should not be increased in treating class i and ii malocclusions . Therefore, prevention of extrusion or active intrusion of the maxillary molars is required to maintain or decrease the mandibular angle . The chi - square test showed an apparent discrepancy in the sample distribution of angle's classification of malocclusion between the groups (p = 0.018). Although the measurements were not compared between skeletal class i and skeletal class ii malocclusions, the differences in the cephalometric variables imply that the skeletal pattern between the groups was different, and the strain group showed a higher tendency for retrognathism, convex profile, and dolichofacial type . Patients showing unfavorable strain at the soft - tissue chin may be managed by an orthodontic intervention - often involving retraction of anterior teeth - to establish satisfactory anterior dental relationship accordingly which induces changes in the lip posture, leading to lip closure without unnecessary tension in the lips and soft - tissue chin, and thus a more desirable profile is obtained.1 recognition of the variables predicting strain of the soft - tissue chin may enable the establishment of a guideline for the important dentoskeletal factors to be considered during diagnosis and treatment planning . To minimize strain of the soft - tissue chin, the treatment modality should be oriented toward decreasing the u1-npog, overjet, and u1-upper lip values while increasing the odi value . Strain of the soft - tissue chin is influenced by not only the underlying dentoskeletal structures but also the thickness, length, and tone of the soft tissue.10 therefore, further studies should include soft - tissue variables to improve the predictability of the soft - tissue profile after orthodontic treatment . Given that the present study was limited to only korean women, studies including men or various age or racial groups are warranted to compare the discrepancies arising from these predictive factors . Odi, u1-npog, overjet, and u1-upper lip were found to be the discriminants for classifying the female adult subjects with class i or ii molar relationships into the no - strain or strain group, and the prediction rate of these variables for the group classification was 81.8%.in the cart model, u1-npog was the most predictive variable for categorizing the subjects into the no - strain or strain group, with a cut - off value of 14.2 mm.the subjects in the strain group showed a retruded mandible, vertical skeletal pattern, convex profile, and protruded dentoalveolar pattern compared with those in the no - strain group . Odi, u1-npog, overjet, and u1-upper lip were found to be the discriminants for classifying the female adult subjects with class i or ii molar relationships into the no - strain or strain group, and the prediction rate of these variables for the group classification was 81.8% . In the cart model, u1-npog was the most predictive variable for categorizing the subjects into the no - strain or strain group, with a cut - off value of 14.2 mm . The subjects in the strain group showed a retruded mandible, vertical skeletal pattern, convex profile, and protruded dentoalveolar pattern compared with those in the no - strain group.
Rheumatoid arthritis (ra) is a chronic, systemic inflammatory disorder that primarily targets the synovium and articular cartilage, leading to synovitis and progressive joint damage . However the pathophysiology of ra is poorly understood, the pro - inflammatory adipocytokines such as leptin, resistin and visfatin have been suggested to have an important role in the pathogenesis of the disease (1). Leptin, which is mainly produced by white adipose tissue (wat) cells, is a hormone encoded by the obese gene (ob), the murine homologue of the human lep (2). In addition to its well - known ability to regulate body weight by inhibiting food intake and increasing energy consumption, it also has inflammatory and immunomodulatory effects (3). Serum leptin levels were increased in ra patients compared to healthy controls (4 - 7); it was significantly higher in women than in men (8). Visfatin, a growth factor for b lymphocyte precursors, is another pro - inflammatory adipocytokine, seen in liver, skeletal muscles and bone marrow and produced by visceral wat, which mimics the effects of insulin (9, 10). Serum visfatin levels were increased in patients with ra (4, 7, 11 - 13). Recently, reports have suggested the significant role of adipocytokines such as visfatin in mediating joint damage (4, 14, 15). Nevertheless, it is still a matter of debate whether infliximab significantly changes the visfatin levels (16). The purpose of this study was to examine the association between serum levels of adipocytokines (leptin and visfatin) and radiographic joint damage in patients with ra . This study was conducted on 29 patients with erosive rheumatoid arthritis (ra) and 25 patients with non - erosive ra . All patients fulfilled the american college of rheumatology classification criteria for ra (17). Eligible patients were those who met all of the following criteria: having ra for at least 1 year, but no more than 5 years; aged between 20 and 50 at the disease onset; and bmi 30 . Patients were excluded if they had any of the following concomitant diseases: erosive joint diseases such as gout, seronegative spondyloarthropathies; osteoarthritis; blood pressure 140/90 mmhg; cardiovascular diseases such as congestive heart failure or coronary artery diseases . Each subject signed a written informed consent after explaining the objective of the study and verification of the inclusion and exclusion criteria . X - rays of the hands and feet were obtained from each of the subjects and 20 joints (the wrists, 1 to 5 metacarpophalangeal joints, and 2 to 5 metatarsophalangeal joints) were evaluated by a particular radiologist who was blinded to the study . The amount of damage was quantified by using the larsen score, with ranging from 0 to 100 for all 20 joints (18). After overnight fasting, blood samples were drawn from all patients and stored at -20c until the time of analysis by leptin kit, (visfatin kit: rav bio human visfatin e1 america; me diagnose company, code: ref - e07 germany) through elisa . Descriptive statistics were calculated as mean standard deviation (sd) or median [interquartile range (iqr)] according to distributions of continuous variables . Statistical analysis was performed with spss for windows software version 11.5 (spss inc ., the mann - whitney test was used to determine the mean differences . For evaluating relationships among normally distributed variables, the pearson correlation coefficient was calculated, whereas the spearman's rho was calculated in case of nonparametric data . Statistical analysis was performed with spss for windows software version 11.5 (spss inc ., chicago, il, usa). Mean values and standard deviations were used for continuous variables . For comparing means, the mann - whitney test was used to determine the mean differences . For evaluating relationships among normally distributed variables, the pearson correlation coefficient was calculated, whereas the spearman's rho was calculated in case of nonparametric data . In our study, age, sex and bmi did not show any statistically significant differences between the two groups of patients (erosive and non - erosive ra) (p - values> 0.05). The median disease durations were 5 years (iqr (3 - 5)) and 2 years (iqr (1 - 4.5)) in patients with erosive and non - erosive ra, respectively; which showed statistically significant differences between the two groups of patients (p=0.003). Leptin concentrations did not show any statistically significant differences between the two groups of patients (p=0.903). Visfatin concentrations were significantly higher in patients with erosive ra (34.6 [29.1 - 100] ng / ml) compared with non - erosive ra (28.4 [22.1 - 34.5] ng / ml) (p=0.013) (table 1). Leptin and visfatin correlations with bmi, disease duration and larsen score are shown in table 2 . Bmi correlated positively with leptin (r=0.494, p<0.001), figure 1 while disease duration correlated positively with visfatin (r=0.488, p<0.001) figure 2 . No correlations were seen between larsen score and leptin and visfatin (r= -0.318, p=0.093 and r=0.254, p=0.184 respectively). By using the linear regression coefficient model, leptin showed correlation with bmi (t=4.096, p<0.001) and visfatin showed positive correlations with c - reactive protein (crp) and disease duration (t=2.165, p=0.035 and t=2.719, p=0.009 respectively). Nevertheless, using univariate variance analysis, there was no correlation between leptin and bmi in the two groups of ra (p=0.508), and leptin serum levels in the two study groups were not significantly different, not considering the bmi (p=0.522). While there was a positive correlation between visfatin and crp (p=0.008), no correlation was seen between visfatin and disease duration in the two erosive and non - erosive ra groups (p=0.247). There are a few studies about the effect of adipokines like leptin and visfatin on radiographic changes of involved joints in rheumatoid arthritis . In giles et al study, in 2009, there was no association between the level of leptin and radiographic joint damage (14). However, in rho et al studies, there was a positive correlation between these adipokines with radiographic changes, and higher levels of leptin were associated with less radiographic damage (13). In this case - control study, we evaluated the association between the serum levels of leptin and visfatin with radiographic damage in two groups of patients with rheumatoid arthritis: with and without radiographic damage, to better understand the physiology of radiologic joint damage in rheumatoid arthritis patients which is the most important cause of morbidity in rheumatoid arthritis . We found that serum levels of visfatin in our rheumatoid arthritis patients with joint erosion were much higher than those in patients without joint erosion . There was no correlation between serum levels of visfatin with either sedimentation rate or crp . There was a statistically significant positive correlation between visfatin serum levels with disease duration (p<0.001, r=0.538). C - reactive protein level and disease duration were correlated to serum visfatin by linear regression method . Univariate analysis showed that serum visfatin in our two study groups had a statistically significant positive correlation (p=0.008) with radiographic damage disregarding crp as co - founder (p=0.008). However, dispensing with the disease duration, visfatin serum levels in the two groups had no statistical significance (p=0.695). These findings represent that serum visfatin level, independently, with serum crp as an inflammatory marker, is associated with joint damage in radiographic studies, but this correlation (visfatin level and radiographic damage) depends on the disease duration . Our study was similar to the rho et al study in 2009, which revealed that visfatin serum levels were positively correlated with radiographic joint damage considering age, gender, disease duration, bmi, and inflammation, with statistical significance (13). Although visfatin is an adipokine, based on previous studies, there is no correlation between visfatin and obesity (17). However, some studies have shown that there is an association between visfatin and inflammatory markers (18, 19). In the rho et al study there was a positive relationship between visfatin serum levels and inflammatory mediators like crp, tnf and il6 with statistical significance, but no correlation between visfatin and bmi . In our study, we could not find a correlation between bmi and visfatin levels . This finding is probably due to the difference between inflammatory markers and also qualitative use of data in our study . Brentano and colleagues reported that visfatin was accumulated in the invasive synovial tissue in rheumatoid arthritis patients and its levels were increased in synovial fibroblasts as well (20). Visfatin is able to induce il6, matrix metalloproteinase (mmp1 and mmp3) in synovial fibroblasts in ra patients and induce tnf and il6 in monocytes too (20). In our study, we found that visfatin level is associated with radiographic damage and has a catabolic effect on joints; therefore it has an important role in pathophysiology in rheumatoid arthritis . Busso et al, revealed that in arthritic rats, visfatin inhibitors induce remission exactly like tnf inhibitors (21). Interestingly, pharmacologic inhibition in visfatin, decreases intracellular nad levels (intracellular nucleotide adenine deaminase) in inflammatory cells and reduces production of il6 and tnf in the involved joints of ra patients (21). With respect to above findings, we can come to the conclusion that visfatin is an independent mediator in the pathogenesis of rheumatoid arthritis and is related to radiographic joint damage . Visfatin level was not related to esr, crp, larsen score or disease duration, but had a meaningful, positive statistical relation with bmi (p=0.004, r=0.385). Our study showed that leptin levels in ra patients with or without joint erosion were not any different . Rho et al (13) showed that serum leptin level associated with less joint space narrowing after suppression of inflammatory process . Also there is a positive relation among leptin, das28, crp, il6, and bmi which is statistically significant in ra patients . In our study, we found a correlation between leptin and bmi and no association between leptin with disease activity, crp and il6 . This discrepancy might be due to the design difference between our study and that of rho et al . We did not evaluate disease activity score in our patients; all patients had had rheumatoid arthritis for less than 5 years . According to the studies of westhoff et al (22), kaufmann et al (23) and van der helmm et al (24) obesity has a protective effect in joint damage in ra patients (22 - 24). Therefore, the important roles of adipocytokines like leptin and adiponectin in obesity should be considered in ra patients . We found that visfatin level as an inflammatory marker, independently of crp level, is associated with joint radiographic damage and is related to disease duration . The mechanism of visfatin as a proinflammatory and its catabolic role is not known and visfatin biology seems to be complex; the key roles of visfatin as a treatment are still obscure . Additionally, our study is one of the earliest in the field of association of adipocytokine levels and radiographic joint damage . Therefore, we suggest evaluating levels of adipocytokines before and after treatment in longitudinal studies to make clear the relationship between adipocytokines and joint damage in human models in the future . In future research, it would be reasonable to measure leptin and visfatin simultaneously, in addition to the other adipocytokines like adipocytokines of resistin.
Chronic kidney disease is a clinical syndrome consisting of a variety of symptoms and metabolic disorders that arise from the progressive and irreversible decline of kidney function . The end stage of chronic kidney disease is uremia [13], which is not an independent kidney disease, but rather a series of clinical manifestations that make uremia a syndrome that is common to most end - stage kidney diseases . During end - stage uremia, for example, uremia patients can experience severe conditions such as heart failure, psychosis, coma, or other life - threatening complications . Uremia can be caused by many factors such as chronic glomerulonephritis, chronic pyelonephritis, renal tuberculosis, renal arteriosclerosis, stones in the urinary tract, enlarged prostate, bladder cancer, lupus, and diabetes . The kidneys can be damaged by various factors that severely affect kidney function, leading to defects in nitrogen metabolism and waste excretion . Excess accumulation of wastes can upset internal balances that in turn cause further damage to the kidneys and other organs . Renal failure can also be caused by extra - renal factors such as heart failure, urinary tract obstruction, and urinary reflux . Uremia results in the bodily retention of many different compounds that can accumulate to dangerous levels . Current research on uremia has mainly focused on the relationship between metabolomics and renal disease, and more than 90 different uremia - related compounds, some of which are toxic, have been identified . However, the exact genetic and/or molecular mechanism of uremia is still not fully understood because of its complex etiology and the difficulty in obtaining tissue samples . Thus, several studies have examined transcriptome expression of blood or urine in uremia, and results from these studies identified many mirnas and genes, as well as related biological functions and pathways, that are important for uremia [1215]. For a more comprehensive understanding of the mechanisms that are involved in uremia, we analyzed a whole - genome microarray case - control study of 75 uremia patients and 20 healthy controls included in the national center for biotechnology information (ncbi) gene expression omnibus (geo), and performed a gene co - expression analysis to construct networks of differentially expressed genes (degs) in uremia . Finally, mirna enrichment analysis was used to detect key mirnas in each hub model . We hope the results from this study may provide a more comprehensive understanding of the molecular mechanisms that are important in uremia . For this study we selected the gene expression profile dataset with accession number gse37171 from the ncbi geo . The dataset was acquired using an affymetrix human genome u133 plus 2.0 array (platform number gpl570) that analyzed peripheral blood from 75 end - stage renal failure (uremia) patients and 20 healthy controls . The raw data were first normalized by robust multi - array average (rma) background subtraction, and quantile normalized using affymetrix expression console software; all background noises were removed . Then we used significance analysis of microarrays (sam) algorithm (\|log fold change\|>1; fdr <0.05) to screened differentially expressed genes (degs) between uremia patients and control subjects . Gene co - expression networks were performed by pearson s correlation with \|r\|> 0.9 and p<0.01 . The co - expressed hub module analysis was performed on the co - expression network using the molecular complex detection (mcode) plugin in cytoscape with a degree cutoff=2, node score cutoff=0.2, k - core=2, and maximum depth=100 . Significant co - expressed modules with mcode score> 4 and more than six nodes were selected . We used the database for annotation, visualization, and integrated discovery (david) to identified the biological processes and/or functions involving the hub modules . One of david s features is a functional annotation cluster tool wherein similar gene ontology (go) categories, based on the parent / child go term associations and the number of shared genes, can be grouped into a functional cluster . We used this feature to identify the relationship between the go terms (biological processes in the go database) and kyoto encyclopedia of genes and genomes (kegg) pathways . The threshold enrichment score for the enriched clusters was arbitrarily set as 1 . In this study the target genes of all mirnas were predicted using five prediction algorithms: pictar, diana - microt, miranda, rna22, and targetscan . We collected 249 mirnas, 5,828 genes, and 60,038 interactions of mirna to gene . The mirna enrichment analysis is a useful tool to reveal modules of target gene sets with expression levels that change significantly relative to the background . The p values of certain mirnas and their target genes was estimated by the cumulative hypergeometric algorithm, and the function is as follows: in which m is the total number of genes tested, n is the number of genes targeted by a certain mirna, and k represents the number of degs in a given module; x represents the number of genes overlap between degs in a given module and genes targeted by a certain mirna . At last, for this study we selected the gene expression profile dataset with accession number gse37171 from the ncbi geo . The dataset was acquired using an affymetrix human genome u133 plus 2.0 array (platform number gpl570) that analyzed peripheral blood from 75 end - stage renal failure (uremia) patients and 20 healthy controls . The raw data were first normalized by robust multi - array average (rma) background subtraction, and quantile normalized using affymetrix expression console software; all background noises were removed . Then we used significance analysis of microarrays (sam) algorithm (\|log fold change\|>1; fdr <0.05) to screened differentially expressed genes (degs) between uremia patients and control subjects . Gene co - expression networks were performed by pearson s correlation with \|r\|> 0.9 and p<0.01 . The co - expressed hub module analysis was performed on the co - expression network using the molecular complex detection (mcode) plugin in cytoscape with a degree cutoff=2, node score cutoff=0.2, k - core=2, and maximum depth=100 . Significant co - expressed modules with mcode score> 4 and more than six nodes were selected . We used the database for annotation, visualization, and integrated discovery (david) to identified the biological processes and/or functions involving the hub modules . One of david s features is a functional annotation cluster tool wherein similar gene ontology (go) categories, based on the parent / child go term associations and the number of shared genes, can be grouped into a functional cluster . We used this feature to identify the relationship between the go terms (biological processes in the go database) and kyoto encyclopedia of genes and genomes (kegg) pathways . In this study the target genes of all mirnas were predicted using five prediction algorithms: pictar, diana - microt, miranda, rna22, and targetscan . We collected 249 mirnas, 5,828 genes, and 60,038 interactions of mirna to gene . The mirna enrichment analysis is a useful tool to reveal modules of target gene sets with expression levels that change significantly relative to the background . The p values of certain mirnas and their target genes was estimated by the cumulative hypergeometric algorithm, and the function is as follows: in which m is the total number of genes tested, n is the number of genes targeted by a certain mirna, and k represents the number of degs in a given module; x represents the number of genes overlap between degs in a given module and genes targeted by a certain mirna . At last, we identified the degs between uremia patients and controls by the sam algorithm (\|log fold change\|> 1; fdr <0.05) and found 1,399 degs, including 311 upregulated genes and 1,088 downregulated genes . To investigate the interrelation between uremia - related degs, co - expression networks analysis was used to construct degs interaction networks in this study . As the results showed in figure 1, we found a total of 1,046 genes (179 and 867 upregulated and downregulated genes, respectively) and 59,290 interactions between the genes (figure 1). The interactions could be grouped into four networks that had more than four nodes, including one network wherein all nodes were downregulated genes (861 nodes, 57,604 interactions) and three networks that had nodes where all genes were upregulated (132 nodes, 1,589 interactions; 32 nodes, 83 interactions; 5 nodes and 5 interactions, respectively). We next performed a co - expressed hub module analysis on the co - expression networks using the molecular complex detection (mcode) plugin in cytoscape (mcode score> 4 and nodes> 6). We also examined the biological processes and/or functions involving the modules by performing functional enrichment and clustering analysis tools in david . This analysis identified nine modules (figure 2, table 1): m1 (score=88.249) containing 221 downregulated degs and 19,503 interactions that were significantly enriched in membrane - enclosed lumen, negative regulation of gene expression, apoptosis, and rna splicing via transesterification reactions; m2 (score=16.976) containing 126 downregulated degs and 2,139 interactions that were significantly enriched in membrane - enclosed lumen, chromosome organization, nucleotide binding, endosome, and golgi apparatus; m3 (score=14.944) containing 36 upregulated degs and 538 interactions that were significantly enriched in membrane - enclosed lumen;m4(score=6.944) containing 72 down degs and 500 interactions that were significantly enriched in protein localization, post - transcriptional regulation of gene expression, rna processing, and hormone secretion; m5 (score=3.778) containing nine upregulated degs and 34 interactions that were significantly enriched in metal ion binding; m6 (score=3.643) containing 14 upregulated degs and 51 interactions that were significantly enriched in induction of apoptosis; m7 (score=3.415) containing 41 downregulated degs and 141 interactions that were significantly enriched in mrna processing and gtpase regulator activity; m8 (score=2.267) containing 30 downregulated degs and 68 interactions that were significantly enriched in protein localization; m9 (score=2.071) containing 14 upregulated degs and 29 interactions that were significantly enriched in membrane - enclosed lumen . Mirna enrichment analysis was used to predict mirnas that could significantly target genes in each module . This analysis clearly showed that 15 mirnas were selectively enriched in three modules (table 2, figure 3, p<0.01). Of these, hsa - mir-505 - 3p, hsa - mir-490 - 3p, hsa - mir-411 - 5p, hsa - mir-212 - 3p, hsa - mir-154 - 5p, and hsa - mir-132 - 3p were significantly enriched for degs in m1, which may indicate that these mirnas are involved in regulating proteolysis, membrane - enclosed lumen, negative regulation of gene expression, negative regulation of gene expression, apoptosis, and rna splicing via transesterification reactions functions by targeting the degs in this module . Furthermore, the mirnas hsa - mir-875 - 5p, hsa - mir-590 - 3p, hsa - mir-448, hsa - mir-381 - 3p, hsa - mir-300, hsa - mir-22 - 3p, hsa - mir-203a, hsa - mir-154 - 5p, and hsa - mir-153 - 3p were significantly enriched in m4 degs, suggesting that these mirnas may regulate posttranscriptional regulation of gene expression, rna processing, and hormone secretion functions . The mirna hsa - mir-139 - 5p was significantly enriched in m8 degs, such that this mirna may affect protein localization functions . Notably, hsa - mir-154 - 5p was significantly enriched with degs in both m1 and m4 . We identified the degs between uremia patients and controls by the sam algorithm (\|log fold change\|> 1; fdr <0.05) and found 1,399 degs, including 311 upregulated genes and 1,088 downregulated genes . To investigate the interrelation between uremia - related degs, co - expression networks analysis was used to construct degs interaction networks in this study . As the results showed in figure 1, we found a total of 1,046 genes (179 and 867 upregulated and downregulated genes, respectively) and 59,290 interactions between the genes (figure 1). The interactions could be grouped into four networks that had more than four nodes, including one network wherein all nodes were downregulated genes (861 nodes, 57,604 interactions) and three networks that had nodes where all genes were upregulated (132 nodes, 1,589 interactions; 32 nodes, 83 interactions; 5 nodes and 5 interactions, respectively). We next performed a co - expressed hub module analysis on the co - expression networks using the molecular complex detection (mcode) plugin in cytoscape (mcode score> 4 and nodes> 6). We also examined the biological processes and/or functions involving the modules by performing functional enrichment and clustering analysis tools in david . This analysis identified nine modules (figure 2, table 1): m1 (score=88.249) containing 221 downregulated degs and 19,503 interactions that were significantly enriched in proteolysis, membrane - enclosed lumen, negative regulation of gene expression, apoptosis, and rna splicing via transesterification reactions; m2 (score=16.976) containing 126 downregulated degs and 2,139 interactions that were significantly enriched in membrane - enclosed lumen, chromosome organization, nucleotide binding, endosome, and golgi apparatus; m3 (score=14.944) containing 36 upregulated degs and 538 interactions that were significantly enriched in membrane - enclosed lumen;m4(score=6.944) containing 72 down degs and 500 interactions that were significantly enriched in protein localization, post - transcriptional regulation of gene expression, rna processing, and hormone secretion; m5 (score=3.778) containing nine upregulated degs and 34 interactions that were significantly enriched in metal ion binding; m6 (score=3.643) containing 14 upregulated degs and 51 interactions that were significantly enriched in induction of apoptosis; m7 (score=3.415) containing 41 downregulated degs and 141 interactions that were significantly enriched in mrna processing and gtpase regulator activity; m8 (score=2.267) containing 30 downregulated degs and 68 interactions that were significantly enriched in protein localization; m9 (score=2.071) containing 14 upregulated degs and 29 interactions that were significantly enriched in membrane - enclosed lumen . Mirna enrichment analysis was used to predict mirnas that could significantly target genes in each module . This analysis clearly showed that 15 mirnas were selectively enriched in three modules (table 2, figure 3, p<0.01). Of these, hsa - mir-505 - 3p, hsa - mir-490 - 3p, hsa - mir-411 - 5p, hsa - mir-212 - 3p, hsa - mir-154 - 5p, and hsa - mir-132 - 3p were significantly enriched for degs in m1, which may indicate that these mirnas are involved in regulating proteolysis, membrane - enclosed lumen, negative regulation of gene expression, negative regulation of gene expression, apoptosis, and rna splicing via transesterification reactions functions by targeting the degs in this module . Furthermore, the mirnas hsa - mir-875 - 5p, hsa - mir-590 - 3p, hsa - mir-448, hsa - mir-381 - 3p, hsa - mir-300, hsa - mir-22 - 3p, hsa - mir-203a, hsa - mir-154 - 5p, and hsa - mir-153 - 3p were significantly enriched in m4 degs, suggesting that these mirnas may regulate protein localization, posttranscriptional regulation of gene expression, rna processing, and hormone secretion functions . The mirna hsa - mir-139 - 5p was significantly enriched in m8 degs, such that this mirna may affect protein localization functions . Notably, hsa - mir-154 - 5p was significantly enriched with degs in both m1 and m4 . Gene co - expression network analysis is a useful tool in genomics studies, because of its extraction of genes / mirnas with similar expression patterns from high throughput molecule expression datasets . The analysis could identify the relationships between genes and facilitated the understanding of the regulatory mechanism of genes . In the present study, we used the degs to construct the gene co - expression networks, which not only could identify the gene interactions / networks related to uremia, but also could reduce the false positive rate of the degs . Meanwhile, the hub module analysis of differentially expressed genes identified clusters of genes with similar expression profiles that are suggestive of co - regulation . Exploration of these co - regulated gene modules could help find underlying regulation molecules that are pivotal for gene expression in uremia . To explore the molecular mechanisms of degs in uremia, mirna enrichment analysis was used to predict pivotal mirnas that could significantly target genes in each functional module . Results from these bioinformatics studies might inform future studies of the pathways and mechanisms that could be targeted for uremia treatments . Recently, some reports have explored the molecule alterations in the blood of ckd patients [2831]. . Found that over one third of human genes were differential expressed in blood of uremia, and involved in various biological functions and pathways, including many immune and biological mechanisms . Meanwhile, granata et al . Screened genome expression in peripheral blood mononuclear cells of ckd patients, and found some degs involved in mitochondrial respiratory system . Moreover, neal et al . Found the mass of mirnas were reduced in the blood of ckd patients . All these reports indicated a profound magnitude of the changes in ckd, but not enough to define further critical molecular mechanisms in ckd . However, the bioinformatics tools used in our study might provide us with insight into critical biological alterations . In this study, co - expression analysis and co - expressed hub module analysis found nine hub modules that may play important roles in uremia . For example, module m1 was significantly enriched in proteolysis, membrane - enclosed lumen, negative regulation of gene expression, apoptosis, and rna splicing via transesterification reactions . These biological functions have been demonstrated to have significant roles in uremia [3235]. In accord with the clinical characteristics of uremia, protein fragments in plasma and urine are a hallmark of many renal diseases, and can be an important component of plasma and urine for diagnostic and research purposes . Meanwhile, uremia is typically accompanied by apoptotic erythrocyte death that may lead to anemia ., posttranscriptional regulation of gene expression, rna processing, and hormone secretion functions, suggesting that in uremia homeostasis may be severely affected and accompanied by significant disturbances in rna and protein metabolism . Notably, the membrane - enclosed lumen function was significantly enriched in modules m1, m2, m3, and m9 . Indeed, uremia is typically accompanied by changes in cellular homeostasis that involve metabolic disturbances in membrane - enclosed lumens such as the nucleus and endoplasmic reticulum . Micrornas are small non - coding, single - stranded rnas that regulate messenger rnas at the post - transcriptional level . The role of mirnas in chronic kidney disease (ckd) has recently gained increased attention . In this study, we found 15 key mirnas that were significantly enriched in three co - expressed modules . Of these mirnas, mirna-21 - 3p and mirna-210 - 3p were shown to be significantly related to uremia . Moreover, hsa - mir-154 - 5p was significantly enriched in both m1 and m4 degs . However, the other mirnas identified in this study await further characterization and investigation for their role in uremia - related mechanisms . Our study provides a comprehensive perspective of gene expression in uremia using a combination of gene co - expression network analyses and degs analyses to identify nine hub co - expressed modules that can be assigned to corresponding biological functions . The 15 key mirnas identified in the mirna enrichment analysis likely also play important roles in uremia . Further study of the networks and mirnas identified in this study may allow a better connection of biological functions, genes, and mirnas that underpin the network modules, and in turn could be used to elucidate the molecular mechanisms involved in uremia . In conclusion, our study provided an orientation for further work, and the bioinformatics analysis pipeline of this study provides a model for others.
Mccune albright syndrome is rare with an estimated prevalence of 1 in 100,000 to 1 in 1,000,000 persons . The classical clinical triad consists of fibrous dysplasia of the bone, caf - au - lait skin spots and precocious puberty . She had a short stature, round face, thick neck, and short fourth metacarpals and metatarsals . Serum calcium, phosphorus and parathyroid concentrations were normal, but gonadotropin hormones were very low . X - ray examination revealed short fourth and fifth metacarpals, short left metatarsal, and short fibula . These local bony abnormalities along with the biochemical findings helped us to diagnose this case as an unusual presentation of primary hypogonadism with features of mccune albright s syndrome where there was amenorrhea rather than preocious puberty . It is commonly seen in patients with albright s hereditary osteodystrophy (aho) which is a constellation of physical features that include short adult stature, obesity, brachydactyly, and ectopic ossifications . Aho patients who have normal end - organ responses to parathyroid hormone (pth) constitute the rare disorder pseudopseudohypoparathyroidism (pphp). A short fourth metacarpal is seen in approximately 65% of such patients (2). This is distinct from pseudopseudohypoparathyroidism (pphp) which presents with hypocalcemia, hyperphosphatemia, and high levels of parathyroid hormone (pth). It is associated with a possible resistance toward several hormones such as thyroid stimulating hormone (tsh), luteinizing hormone (lh), follicle stimulating hormone (fsh), growth hormone releasing hormone (ghrh) that mediate their action through the g - protein - coupled receptors (3). An eighteen - year old female with height of 138 cm and weight of 66 kg was referred to the endocrine department of a multispecialty hospital of eastern india, in mid 2015 . However, the reported menarche was actually an episode of induced bleeding due to an unknown medication provided by a local doctor . Born to nonconsanguineous parents, the patient was an unmarried college student who reported to be apparently healthy with no significant past history . There was no complaint of symptoms such as cramping, tetany, twitching, or seizure . There was no history of thyroid or other surgery, radiation or any systemic illness . Her blood pressure was 100/60 mmhg, temperature 37c, pulse rate 78 min and respiratory rate 20 min . Physical examination revealed breast bud formation with a small area of surrounding glandular tissue and areola beginning to widen . The fourth and fifth metacarpals on both sides were short with the knuckles being depressed in the clenched fist position (figure 1). Chvostek s sign, trousseau s sign, and signs of neuromuscular irritability were negative . Depressed 4th and 5th finger knuckles in the clenching fist position when a family history was sought, the patient revealed she felt that her grandmother had the same general physical features of short height, round face, thick neck and particularly the appearance of her hands and feet . X ray of the skull (figure 3) revealed normal appearance of the pituitary fossa . Shortening of metacarpals iv and v x ray of the skull showing normal pituitary fossa reports from tests done six months back in an outside laboratory revealed lh 0.8 miu / ml (1.58), fsh 2 miu / ml (312), prolactin 4.1 ng / ml (535), and dehydroepiandrosterone sulphate (dheas) 0.79 g / dl (25.9460.2). At presentation to our hospital, laboratory investigations were repeated . The fasting plasma glucose, serum calcium, phosphorus and alkaline phosphatase, blood urea, blood creatinine, serum 25 hydroxy vitamin d, serum magnesium, 24 hr urinary calcium, parathormone (pth), tsh, growth hormone (gh), insulin- like growth factor 1 (igf-1) levels and erythrocyte sedimentation rate (esr) were within the reference ranges . Nmol / l (> 300), estradiol <9 pg / ml (18575), and fsh 1.63 miu / ml (312). Chromosomal karyotyping revealed normal female karyotype (46xx) with no numerical or structural chromosomal anomalies at 450550 banding resolution . Due to financial constraints of the patient, mri of the hypophysis, though advised, could not be done . Molecular genetic testing done at another laboratory outside the hospital identified a mutation in the gnas gene . This case has the classic findings of albright s facies with short fourth metacarpals and metatarsals, normal serum calcium, phosphate and pth levels diagnostic of pseudopseudohypoparathyroidism with an added unusual feature of hypogonadism . (4) described the metacarpal sign (shortening of the fourth and fifth digits, presenting as dimpling over the knuckles of a clenched fist) as a diagnostic marker of gonadal dysgenesis . In males, this metacarpal sign occurs much more frequently in the presence of some gonadal anomaly than when the gonads are normal . Patients showing the metacarpal sign usually have delayed skeletal development, reflected as short height and short fourth and fifth metacarpals . This deformity is seen more often in females and is hereditary in nature (5). Short fourth and fifth metacarpals and metatarsals are seen in pseudo - pseudohypoparathyroidism, gonadal dysgenesis (turner s syndrome / chromosomal disorder 45xo), pseudohypoparathyroidism and familial brachydactylism, with hypogonadism being associated with the first two conditions (6). Trauma is the most common cause of this deformity but other causes include neurofibromatosis, congenital adrenal hyperplasia due to 11 beta hydroxylase deficiency, (5) familial short stature, hereditary multiple exostoses, patients with homocystinuria and other less common syndromes (8). An alteration in the coding sequence of the gnas gene leads to a haplo - insufficiency and a dysmorphic phenotype referred to as albright s syndrome or albright s hereditary osteodystrophy (aho). It is a clinical syndrome defined by specific physical features that include short stature, obesity, round - shaped face, subcutaneous ossifications and brachytherapy mainly of the fourth and fifth metacarpals . Patients with mutations on their maternally derived allele are likely to have associated phpia, whereas mutations on the paternal allele usually cause pphp . Isolated pth resistance (phpib) can result from mutations within the gnas1 gene but is more commonly caused by epigenetic imprinting abnormalities affecting the upstream exon 1a (9). In php, there is a hormonal resistance to pth at the kidney level and to tsh at the thyroid level while in pphp, there are few clinical signs with no hormonal resistance (10). However, according to a recent publication, pth and other hormone resistance may be seen in both disorders (11). The characteristic physical features of short stature, round face, brachydactyly of the fourth and fifth metacarpals and the biochemical findings of normal serum calcium, phosphorus and parathyroid hormone concentrations along with a very low level of gonadotropin hormones led us to the diagnosis of albright s syndrome in this patient who presented to us with primary amenorrhea and hypogonadism.
The cytokine interferon- is critical for protection against viral and bacterial infections and tumor development . Its biological activities require the phosphorylation of stat1 at a single tyrosine residue (stark and darnell, 2012). Stat activation, as it transforms the stat1 dimers into dna binding transcription factors . Stat1 inactivation, namely the enzymatic reversal of tyrosine phosphorylation, accordingly is equally important for physiological signaling (liu et al ., 2011). The tyrosine phosphatase tc45 is the major stat1-inactivating enzyme (ten hoeve et al ., therefore is required for understanding the physiological regulation of ifn signaling as well as for the development of therapeutic stat1 modulators, e.g., for viral and immune diseases (borden et al ., 2007). In the cell nucleus, stat1 inactivation is ultimately limited by the kinetics of dna binding, whereby stat1 is available for dephosphorylation only in its dna unbound state (meyer et al ., 2003). Recent results indicate that dephosphorylation is a multistep process that requires stat1 dimers to undergo extensive spatial reorientation (zhong et al ., 2005; mertens et al ., 2006) hydrodynamic modeling of analytical ultracentrifugation results obtained with purified stat1 indicated moreover that the reorientation of the recombinant stat1 dimers is considerably slower (t1/2 2040 min; wenta et al ., 2008) than the dephosphorylation of endogenous stat1 in living cells (t1/2 <15 min; haspel et al ., 1996). In fact, the acetylation of two particular lysine residues of stat1 was reported to enhance its dephosphorylation by facilitating recruitment of tyrosine phosphatase tc45 (krmer et al ., 2009), but this claim was subsequently invalidated (antunes et al ., 2011). Another posttranslational modification, namely sumo conjugation, can enhance the dephosphorylation of stat1 by increasing its solubility, yet sumo does not itself partake in the actual dephosphorylation step (droescher et al ., 2011). The only stat1-interacting protein known to directly enhance the dephosphorylation reaction thus is -arrestin1 (mo et al ., 2008). The -arrestins are two ubiquitous proteins that are best known for their role as cytoplasmic adapters in the regulation of g protein - coupled receptors and other signaling molecules (dewire et al ., 2007). Additional functions for -arrestins in the nucleus have also been described (kang et al ., 2005). Propose a model whereby -arrestin1, but not -arrestin2, promotes the dephosphorylation of nuclear stat1 by acting as a scaffold to directly facilitate recruitment of phosphatase tc45 . This made -arrestin1 an interesting object for our studies of stat1 dimer reorientation and its effects on dephosphorylation . Here we present the results of our experiments, which contrary to expectations provide evidence against the reported negative - regulatory role of -arrestin1 in stat1 signaling . At first we wanted to confirm that overexpression of -arrestin1 diminishes ifn-induced transcription of a stat1-dependent reporter gene in hela cells . We used c - terminally green fluorescent protein (gfp)-tagged human -arrestin1 and n - terminally flag - tagged rat -arrestin1, which in agreement with its evolutionary conservation can supplant functions and interactions of the human homolog (scott et al ., 2006; both constructs were overexpressed in hela cells but did not diminish ifn-induced reporter gene activity (figures 1a and 1b). We noted that increased -arrestin1 transfection led to an apparent rise in both constitutive and induced reporter gene activity, which overproportionally affected the former . Consequently, when depicted as fold induction ([induced transcription]/[constitutive transcription]), as done by mo et al . (2008), the transcriptional activity indeed appeared to drop with increased -arrestin1 levels (figures 1a and 1b). We identified reduced expression of the -galactosidase control gene as a cause (figure 1c), probably due to titration of limiting coactivators by the cotransfected -arrestin expression vectors . We therefore varied the reporter gene to control gene ratio between 1.5:1 and 25:1 to test whether a different transfection protocol could reveal the reported inhibitory effect of -arrestin1 on stat1-mediated gene induction (figure 1d). However, interferon - inducible reporter gene activity was the same for all reporter - to - control - gene ratios used, and an inhibitory effect of -arrestin1 was not recognizable (figure 1e). Fold induction was likely due to an error associated with the cotransfection of -arrestin1 expression vectors . It was hence not possible for us to rule out a similar error for their published data . Based on the published work, we expected that overexpression of -arrestin1 in hela cells would diminish tyrosine phosphorylation of the endogenous stat1 . However, this was not the case, since stat1 phosphorylation kinetics was identical under all conditions examined regardless of the presence of increased -arrestin1 (figure 2a). To avoid the limitations of transfection assays, we obtained mouse embryonic fibroblasts (mefs) deficient in -arrestin1 and control fibroblasts (kohout et al ., 2001). First, we used a commercially available antibody to confirm -arrestin1 expression in wild - type but not the -arrestin1-deficient mef (figure 2b). We then studied the tyrosine dephosphorylation of stat1 in the wild - type cells and the -arrestin1-deficient cells (figure 2c). This was done by treating the cells for 60 min with ifn before adding the protein kinase inhibitor staurosporine for up to 60 min to terminate abruptly the ongoing protein phosphorylation . As expected, after 1 hr in the presence of staurosporine the concentration of tyrosine - phosphorylated stat1 contrary to the previous report, however, stat1 dephosphorylation was not diminished in the cells lacking -arrestin1 (figure 2c). Thus, stat1 activation kinetics was unchanged regardless of whether -arrestin1 was overexpressed or deleted . We then used -arrestin1-deficient mefs in conjunction with the primer sequences and qpcr conditions of mo et al . (2008) to determine ifn-induced expression of gbp1, cxcl10, and isg15 genes . According to their work interferon - induced transcription of these genes is 5- to 25-fold higher for -arrestin1-deficient mefs than for wild - type cells . In our hands, in contrast, lack of -arrestin1 did not result in increased transcription . In fact, transcription was impaired in the -arrestin1-deficient cells, an observation we also made for the five ifn-inducible genes we tested in addition (figure 3). It remains to be determined if -arrestin1 has a stat1-independent stimulatory role specifically in interferon signaling, or whether the observed effects on gene transcription rather are a reflection of -arrestin1 s involvement in a multitude of cellular functions . These cells are transiently transfected to reconstitute -arrestin1 expression (mo et al ., 2008). We question the appropriateness of this experimental setup to discern the biological activities of individual -arrestins . For example, the authors presume comparable -arrestin1 expression in wild - type cells and their reconstituted mefs based on western blotting results (see their figure 4c). Yet this experiment does not fully clarify the issue in the absence of information about transfection efficiencies and hence cellular expression levels . If, however, the authors presumption is indeed correct, the problem arises that gene expression is upregulated 5- to 25-fold in the arrestin1/2 double - deficient mefs, whereas reconstitution with -arrestin1 merely halves gene expression according to mo et al . This discrepancy indicates that -arrestin double knockout mefs do not revert to wild - type characteristics with regard to stat1 signaling upon transfection of -arrestin1 . As this limits the value of -arrestion1/2 doubly deficient cells for studying the effects specifically of -arrestin1 on stat1, we have instead used cells deficient solely in -arrestin1 . In the final set of experiments flag - tagged stat1 and gfp - tagged -arrestin1 were expressed in hek293 t cells either alone or together . The interaction of -arrestin1 and stat1, which was reported to be maximal in ifn-treated cells, was then analyzed by anti - flag immunoprecipitation . Alternatively, stat1-flag was coexpressed with gfp - tagged stat1 as a positive control since stat1 forms equally stable homodimers before and after stimulation with ifn (wenta et al ., as shown in figure 4a, top panel, probing with anti - flag antibody of extracts from cells cotransfected with flag - tagged stat1 and gfp - tagged--arrestin1 (lanes 1 and 2) or gfp - tagged stat1 (lanes 5 and 6), respectively, showed an 98 kda band expected for flag - tagged stat1 . Reprobing with anti - gfp antibody (middle panel) revealed a band of 80 kda band in lanes 1 and 2 expected for gfp - tagged -arrestin1; and a band of 110 kda in lanes 5 and 6 expected for gfp - tagged stat1 . Probing with anti--arrestin1 antibody (bottom panel) labeled an 80 kda band in lanes 1 and 2, but not 5 and 6, confirming -arrestin1 overexpression . These results demonstrated that both stat1 and -arrestin1 were well - expressed in the cotransfected cells . When the material that coprecipitated with flag - tagged stat1 was probed with anti - gfp to detect coprecipitating -arrestin1-gfp, shown in the middle panel, lanes 3 and 4, a band that comigrated with gfp - tagged--arrestin1 reprobing with anti--arrestin1 antibody nonetheless confirmed this band to contain -arrestin1 (bottom panel). Since the same antibody was used for detection of -arrestin1 and stat1, namely anti - gfp, it was possible to provide also a quantitative assessment for the efficiency with which these two proteins coprecipitated with the flag - tagged stat1 . The quantitative comparison, which was done after normalization of the signals for precipitated -arrestin1 and stat1 using their respective inputs, demonstrated that the interaction of stat1 with itself was three to six times stronger than with -arrestin1 . In our experiments the signal intensities for coprecipitating -arrestin1 were not only weak but moreover did not increase when extracts from ifn-treated cells were used (middle and bottom panels, compare lanes 3 and 4). As an additional control for the specificity of the observed interaction presumably between -arrestin1 and stat1, we performed immunoprecipitations using anti - flag - tag antibody in the absence and presence of its antigen, namely flag - tagged stat1 (figure 4b). As seen in the previous experiments, anti - flag - tag immunoprecipitations with extracts that contained both -arrestin1 and the flag - tagged stat1 gave a weak -arrestin1 signal (middle and bottom panels, lane 7). This signal was expectedly missing when -arrestin1 was missing in the extracts (middle and bottom panels, compare lane 7 to lane 6). However, the converse experiment using extracts devoid of flag - tagged stat1 did not result in absent or at least diminished precipitation of -arrestin1 (middle and bottom panels, compare lanes 7 and 8). We therefore concluded that the observed precipitation of -arrestin1 was not due to interactions specifically with stat1 . Stat1 activation is indispensable for the execution of interferon- functions such as antimicrobial protection . Interacting proteins that can reduce the activity of stat1 are therefore of pharmaceutical interest and potentially of clinical relevance, as was suggested for -arrestin1 by mo et al . However, our experiments did not identify -arrestin1 as a stat1-interacting protein, and they showed that -arrestin1 did not reduce the activation of stat1 . We accordingly found that the transcription of interferon- target genes was not inhibited in the presence of -arrestin1 . In summary, our data do not support a negative - regulatory role for -arrestin1 in interferon- signaling . Immortalized mefs deficient in -arrestin1 and wild - type control cells were provided by dr . T, hela, and mef cells were kept in growth medium, dmem supplemented with 10% fbs and 1% penicillin / streptomycin, in a humidified incubator with 5% co2 at 37c . Cells grown to 70%90% confluence were transfected using lipofectamine according to the manufacturer s recommendations (invitrogen), followed by subsequent experimentation after 24 hr . Plasmids encoding human -arrestin1-gfp (arrb1-gfp) and rat flag--arrestin1 (flag - arrb1) were provided by dr . The interferon--dependent luciferase reporter gene 3xly6e gas - luc containing a triple stat1 binding site was described (wen et al ., 1995). Human (#407306) and mouse ifn (#407303) were obtained from merck millipore . Hela cells were transfected on 24-well plates with 0.25 g / well plasmid encoding -galactosidase, 0.25 g / well 3xly6e gas - luc reporter gene, and the indicated amounts of plasmid encoding -arrestin1 tagged with either gfp or flag . The amount of transfected dna was kept constant by adding plasmid pbluescript (pbs). After 24 hr, the cells were left untreated or treated for 6 hr with 50 u / ml human ifn, followed by cell lysis in buffer containing 25 mm glycylglycine, 15 mm mgso4, 4 mm egta, 1% triton x-100, 1 mm dtt (ph 7.8). Luciferase activity was determined with luciferase - assay - system (promega) according to the manufacturer s instructions . -galactosidase activity was photometrically measured at room temperature at a wavelength of 405 nm after incubation (3060 min) of the extracts (5 l) in 280 l buffer containing 0.75 mm nah2po4/na2hpo (ph 7.4), 1 mm mgcl2, 1 mg / ml o - nitrophenyl--d - galactoside . Control experiments (figure 1d) with varying ratios of luciferase and -galactosidase expression plasmids were done with hela cells under the conditions described before . The [luciferase:gal] ratios stated were obtained by using the following combinations of plasmid 3xly6e gas - luc and psv-gal: [1.5:1] 0.3 and 0.2 g / well, [5:1] 0.42 and 0.08 g / well, [10:1] 0.495 and 0.05 g / well, and [25:1] 0.5 and 0.02 g / well . In addition, a fixed amount (0.25 g / well) of plasmid encoding -arrestin1 (arrb1-gfp) or pbs was included as indicated . The control experiments of figure 1c were done with hela cells on 12-well plates cotransfected with 0.2 g / well of plasmid psv-gal and the indicated amounts per well of flag - arrb1 . Stat1 phosphorylation as shown in figure 2a was determined using hela cells on 6-well plates transfected with 0.8 g / well of the indicated vectors expressing -arrestin1 or -galactosidase as the control . Twenty - four hours later the cells were passaged (dilution 1:2.5), and after a further 24 hr the cells were left untreated or treated for 1 hr with 50 u / ml human ifn, followed by a medium change and incubation with ifn - free growth medium for the indicated time periods . Stat1 dephosphorylation (figure 2c) was detected using wild - type and -arrestin1-deficient mefs left untreated or treated with 50 u / ml mouse ifn for 1 hr . Subsequently, 0.5 m tyrosine kinase inhibitor staurosporine (#569397, calbiochem) was added to the cells for the indicated lengths of time, followed by cell lysis in boiling sds sample buffer . Cells were extracted on ice for 30 min in buffer containing 2 mm egta, 0.2 mm edta, 1 mm na - vanadate, 50 mm naf, 280 mm nacl, 0.5% (v / v) np-40, 10% (v / v) glycerol, 1 mm dtt, protease inhibitors pmsf (0.54 mm) and complete (roche), and 50 mm tris / hcl (ph 7.4). Soluble whole - cell extracts were resolved by sds - page and were analyzed by quantitative western blotting using a li - cor odyssey system as described (droescher et al ., 2011). Anti - mouse stat1 (sc-591), anti - human stat1 (sc-345), anti - gfp (sc-8334), and anti--arrestin1 (sc-9182) were purchased from santa cruz biotechnology . Anti--actin (a5441) and anti - flag m2 (f3165) were from sigma . Anti - phospho - tyr701 stat1 (#9171) was from cell signaling technology; anti--galactosidase (a11132) was from molecular probes . Irdye800cw - conjugated anti - mouse (#926 - 32212) and anti - rabbit (#926 - 32213) igg secondary antibodies were purchased from li - cor bioscience . T cells grown on 10 cm plates were transfected with 2.5 g each of plasmids encoding stat1-flag and -arrestin - gfp or stat1-gfp . Twenty - four hours later cell cultures were passaged (dilution 1:2), and after a further 24 hr the cells were left untreated or treated for 1 hr with 50 u / ml ifn. Soluble whole - cell extracts, containing 500 g protein as determined by bradford assay (biorad), were rotated at 4c with 5 l anti - flag m2 for 90 min before adding 12 l (settled volume) protein a / g plus agarose (sc-2003, santa cruz) and rotating for another 2 hr . After centrifugation (1,000 g) at 4c for 4 min, the precipitate was washed twice with whole - cell extraction buffer and eluted in sds sample buffer for western blotting . Mef cells were grown overnight in serum - depleted growth medium (1% (v / v) fbs), followed by incubation in growth medium with or without 100 u / ml mouse ifn. Subsequently, total rna was extracted, followed by reverse transcription, and expression of selected genes was measured by qpcr using sybr green . The primers used for gbp1, cxcl10, and isg15 were as described by mo et al . Expression of psmb9 was determined with primer pair 5-cgggggtgtcgtggtgggctctg and 5-cgccggcactcctcaggggtcat (reverse), and that of irf9 with 5-gcctttgccccatccccatctc and 5-cccctggccctggaagtactgg (reverse). For further experimental details and the primers for cxcl9, irf1, and icam1,
Case - patients and controls were recruited from jeroen bosch hospital in s - hertogenbosch and bernhoven hospital in oss and veghel; both are regional hospitals located in the center of the q fever epidemic area in the netherlands . The study design was approved by the medical research ethics committee of the university medical centre utrecht . We used existing datasets and spontaneous notifications from the 2 hospitals to identify all chronic q fever diagnoses among patients> 18 years of age during january 1, 2007may 1, 2011 . In the past, chronic q fever is considered proven if c. burnetii is detected by pcr in blood or tissue in the absence of acute infection, but sensitivity of this technique is only 50% (15,16). Persisting high levels of igg to phase i antigens (phase i igg) and, to a lesser extent, phase ii antigens (phase ii igg) are also indicative of chronic q fever (1). The optimal immunofluorescence assay (ifa) cutoff value for phase i igg titer is still matter of debate and is dependent on the test used but is probably within the range of 8001,600 (7,1719). Recently, the dutch q fever consensus group proposed a new diagnostic approach that combines pcr, serologic testing, and clinical data and categorizes cases into proven, probable, or possible chronic q fever (20). Proven cases are those among patients with positive pcr results for c. burnetii in blood or tissue or a phase i igg titer of> 1,024 in combination with a vascular infection proven by positron emission tomography (pet), computed tomography (ct), magnetic resonance imaging (mri), or endocardial involvement according to the major criteria of the modified duke criteria on echocardiogram (21). Probable cases are those among patients with phase i igg titers of> 1,024 and known risk factors: nonmajor valvulopathy according to the modified duke criteria (21), suspected nonvascular or noncardial localization of chronic q fever infection, or aspecific signs of chronic infection . Possible cases are those among patients with phase i igg titers of> 1,024 without other risk factors as listed for probable or proven chronic q fever . In contrast to the other 2 subgroups, in general, possible chronic q fever patients do not receive long - term antimicrobial drug treatment but instead enter a follow - up program; many demonstrate spontaneous decline in phase i igg titers . We defined cases according to these definitions (20) (table 1). * ifa, immunofluorescence assay; pet, positron emission tomography; ct, computed tomography; mri, magnetic resonance imaging . Controls were selected from an existing cohort of patients with acute q fever, seen by general practitioners in 2009, who had positive pcr results for c. burnetii in serum samples . Controls were included if they were> 18 years of age at the time of acute q fever and if the serologic profile was not suggestive of chronic q fever during> 1 year of follow - up (i.e., decreasing antibody titers and phase i igg titer <1,024). Patients with serologic follow - up of <1 year after the episode of acute q fever were excluded from analysis . All case - patients except 1 and all controls lived in the same postal code area (50005400) in the netherlands . Microbiological diagnostics for chronic q fever case - patients consisted of ifa (focus diagnostics, inc ., cypress, ca, usa) of serum samples and pcr for c. burnetii dna in serum, plasma, and tissue samples . The diagnostic workup to evaluate c. burnetii infection in control patients with documented acute q fever had been performed according to a diagnostic algorithm for acute q fever introduced in may 2009 . In brief, serum samples were screened with elisa for igm against c. burnetii phase ii antigens (mii - screen; institut virion serion gmbh, wrzburg, germany). Depending on date of onset of disease and inpatient or outpatient setting, pcr for c. burnetii dna was performed if the mii - screen result was negative (2325). In patients with confirmed acute q fever, serologic follow - up was performed at 3, 6, and 12 months, consisting of ifa for igm and igg against c. burnetii phase i and phase ii antigens . We collected patient characteristics including demographic variables, medical history, medication, pathology and microbiology results, imaging records, therapy, and outcome for case - patients and controls . Case - patient information was already available in the hospital registration systems and was interpreted by 2 researchers (l.k . And s.d . ). All controls were sent a questionnaire and an informed consent form that asked for permission to request patient s data from the general practitioner and from the hospital registration system . Although debatable, routine echocardiographic screening after diagnosis of acute q fever is not the standard of care in the netherlands because no benefit was found in an earlier evaluation (26,27). Therefore, for chronic q fever case - patients and acute q fever controls, details about cardiac valvulopathy were retrieved by review of medical records . Spss version 18.0 was used for storage and analysis of the collected data (spss inc .,, we conducted 3 analyses: 1) an overall analysis of all chronic q fever cases (i.e., proven, probable, and possible); 2) an analysis of proven and probable chronic q fever cases; and 3) an analysis of proven chronic q fever cases only . We performed these analyses to determine whether exclusion of possible chronic q fever and, to a lesser extent, probable chronic q fever (the groups in which disease status is doubtful) influenced the overall results . Univariate and subsequent multivariate logistic regression analyses were performed to calculate odds ratios (ors), corresponding 95% cis, and p values for the development of chronic q fever . In univariate analysis, variables with no observations among case - patients and <2 observations in the control group (or vice versa) were excluded (i.e., hematologic malignancies, bone marrow transplantation, dialysis, renal transplant, nonrenal organ transplant, congenital cardiac deviation, pulmonary diseases, and autoimmune disorder). For potential dichotomous risk factors, i.e., those that had 0 observations among either the case - patients or controls but> 2 observations in the other, we applied a fisher exact test to calculate p values . Variables with> 1 observations and <25% missing values in case - patients and controls, a p value of <0.10 in univariate analysis, or known association in previous reports with the development of chronic q fever were subsequently analyzed in a multivariate model . The variables vascular history and valvulopathy were not included in multivariate analysis because they were included in variables that were listed separately (i.e., vascular prosthesis, aneurysm, other vascular surgery, peripheral arterial disease, cerebrovascular disease, valvular surgery, and nonsurgical valvular disease). Eighteen case - patients and 0 controls had a history of valvular surgery . Because of the expected importance of this risk factor and the high incidence among case - patients, we considered its inclusion in the multivariate analysis critical; moreover, the logistic regression model could not be fitted with this variable excluded . Therefore, we randomly changed one of the observations of the control group from 0 to 1, which artificially reduced the association but enabled us to fit the regression model . The variables age, vascular history, vascular prosthesis, aneurysm, other vascular surgery, cerebrovascular disease, peripheral vascular disease, valvulopathy, valvular surgery, valvular deviation, ischemic heart disease, other cardiovascular diseases, hypertension, dyslipidemia, diabetes, nonhematologic malignancy, and renal insufficiency could be included in multivariate analysis of all groups . The variable immune disorder was also included in multivariate analysis for the probable and proven and the proven subgroups . Differences between case - patients and controls were shown in use of statins, clopidogrel, acenocoumarol, and proton pump inhibitors and hospitalization and adequate treatment during acute q fever (p<0.10). However, these variables could not be included in the multivariate analysis because> 25% of values were missing, most among case - patients in whom an acute q fever episode had gone unrecognized . After selecting predictors for our final multivariate model interactions were not significant and therefore not included in the model . To assess the goodness - of - fit of the final model, we plotted sensitivity and specificity by using a receiver operating characteristic curve and estimated the area under the curve (c - statistic). Case - patients and controls were recruited from jeroen bosch hospital in s - hertogenbosch and bernhoven hospital in oss and veghel; both are regional hospitals located in the center of the q fever epidemic area in the netherlands . The study design was approved by the medical research ethics committee of the university medical centre utrecht . We used existing datasets and spontaneous notifications from the 2 hospitals to identify all chronic q fever diagnoses among patients> 18 years of age during january 1, 2007may 1, 2011 . In the past, chronic q fever is considered proven if c. burnetii is detected by pcr in blood or tissue in the absence of acute infection, but sensitivity of this technique is only 50% (15,16). Persisting high levels of igg to phase i antigens (phase i igg) and, to a lesser extent, phase ii antigens (phase ii igg) are also indicative of chronic q fever (1). The optimal immunofluorescence assay (ifa) cutoff value for phase i igg titer is still matter of debate and is dependent on the test used but is probably within the range of 8001,600 (7,1719). Recently, the dutch q fever consensus group proposed a new diagnostic approach that combines pcr, serologic testing, and clinical data and categorizes cases into proven, probable, or possible chronic q fever (20). Proven cases are those among patients with positive pcr results for c. burnetii in blood or tissue or a phase i igg titer of> 1,024 in combination with a vascular infection proven by positron emission tomography (pet), computed tomography (ct), magnetic resonance imaging (mri), or endocardial involvement according to the major criteria of the modified duke criteria on echocardiogram (21). Probable cases are those among patients with phase i igg titers of> 1,024 and known risk factors: nonmajor valvulopathy according to the modified duke criteria (21), suspected nonvascular or noncardial localization of chronic q fever infection, or aspecific signs of chronic infection . Possible cases are those among patients with phase i igg titers of> 1,024 without other risk factors as listed for probable or proven chronic q fever . In contrast to the other 2 subgroups, in general, possible chronic q fever patients do not receive long - term antimicrobial drug treatment but instead enter a follow - up program; many demonstrate spontaneous decline in phase i igg titers . We defined cases according to these definitions (20) (table 1). * described in (20). Ifa, immunofluorescence assay; pet, positron emission tomography; ct, computed tomography; mri, magnetic resonance imaging . Controls were selected from an existing cohort of patients with acute q fever, seen by general practitioners in 2009, who had positive pcr results for c. burnetii in serum samples . Controls were included if they were> 18 years of age at the time of acute q fever and if the serologic profile was not suggestive of chronic q fever during> 1 year of follow - up (i.e., decreasing antibody titers and phase i igg titer <1,024). Patients with serologic follow - up of <1 year after the episode of acute q fever were excluded from analysis . All case - patients except 1 and all controls lived in the same postal code area (50005400) in the netherlands . Microbiological diagnostics for chronic q fever case - patients consisted of ifa (focus diagnostics, inc ., cypress, ca, usa) of serum samples and pcr for c. burnetii dna in serum, plasma, and tissue samples . The diagnostic workup to evaluate c. burnetii infection in control patients with documented acute q fever had been performed according to a diagnostic algorithm for acute q fever introduced in may 2009 . In brief, serum samples were screened with elisa for igm against c. burnetii phase ii antigens (mii - screen; institut virion serion gmbh, wrzburg, germany). Depending on date of onset of disease and inpatient or outpatient setting, pcr for c. burnetii dna was performed if the mii - screen result was negative (2325). In patients with confirmed acute q fever, serologic follow - up was performed at 3, 6, and 12 months, consisting of ifa for igm and igg against c. burnetii phase i and phase ii antigens . We collected patient characteristics including demographic variables, medical history, medication, pathology and microbiology results, imaging records, therapy, and outcome for case - patients and controls . Case - patient information was already available in the hospital registration systems and was interpreted by 2 researchers (l.k . And s.d . ). All controls were sent a questionnaire and an informed consent form that asked for permission to request patient s data from the general practitioner and from the hospital registration system . Although debatable, routine echocardiographic screening after diagnosis of acute q fever is not the standard of care in the netherlands because no benefit was found in an earlier evaluation (26,27). Therefore, for chronic q fever case - patients and acute q fever controls, details about cardiac valvulopathy were retrieved by review of medical records . Spss version 18.0 was used for storage and analysis of the collected data (spss inc ., within this study, we conducted 3 analyses: 1) an overall analysis of all chronic q fever cases (i.e., proven, probable, and possible); 2) an analysis of proven and probable chronic q fever cases; and 3) an analysis of proven chronic q fever cases only . We performed these analyses to determine whether exclusion of possible chronic q fever and, to a lesser extent, probable chronic q fever (the groups in which disease status is doubtful) influenced the overall results . Univariate and subsequent multivariate logistic regression analyses were performed to calculate odds ratios (ors), corresponding 95% cis, and p values for the development of chronic q fever . In univariate analysis, variables with no observations among case - patients and <2 observations in the control group (or vice versa) were excluded (i.e., hematologic malignancies, bone marrow transplantation, dialysis, renal transplant, nonrenal organ transplant, congenital cardiac deviation, pulmonary diseases, and autoimmune disorder). For potential dichotomous risk factors, i.e., those that had 0 observations among either the case - patients or controls but> 2 observations in the other, we applied a fisher exact test to calculate p values . Variables with> 1 observations and <25% missing values in case - patients and controls, a p value of <0.10 in univariate analysis, or known association in previous reports with the development of chronic q fever were subsequently analyzed in a multivariate model . The variables vascular history and valvulopathy were not included in multivariate analysis because they were included in variables that were listed separately (i.e., vascular prosthesis, aneurysm, other vascular surgery, peripheral arterial disease, cerebrovascular disease, valvular surgery, and nonsurgical valvular disease). Because of the expected importance of this risk factor and the high incidence among case - patients, we considered its inclusion in the multivariate analysis critical; moreover, the logistic regression model could not be fitted with this variable excluded . Therefore, we randomly changed one of the observations of the control group from 0 to 1, which artificially reduced the association but enabled us to fit the regression model . The variables age, vascular history, vascular prosthesis, aneurysm, other vascular surgery, cerebrovascular disease, peripheral vascular disease, valvulopathy, valvular surgery, valvular deviation, ischemic heart disease, other cardiovascular diseases, hypertension, dyslipidemia, diabetes, nonhematologic malignancy, and renal insufficiency could be included in multivariate analysis of all groups . The variable immune disorder was also included in multivariate analysis for the probable and proven and the proven subgroups . The variable pacemaker was also included in multivariate analysis for the proven group . Differences between case - patients and controls were shown in use of statins, clopidogrel, acenocoumarol, and proton pump inhibitors and hospitalization and adequate treatment during acute q fever (p<0.10). However, these variables could not be included in the multivariate analysis because> 25% of values were missing, most among case - patients in whom an acute q fever episode had gone unrecognized . After selecting predictors for our final multivariate model interactions were not significant and therefore not included in the model . To assess the goodness - of - fit of the final model, we plotted sensitivity and specificity by using a receiver operating characteristic curve and estimated the area under the curve (c - statistic). We identified 105 case - patients with proven, probable, or possible chronic q fever; 44 (42%) had proven, 28 (27%) probable, and 33 (31%) possible disease . Of the case - patients with proven chronic q fever, 27 (61%) had positive pcr results for c. burnetii in blood only, 5 (11%) in tissue only, 8 (18%) in tissue and blood, and 4 (9%) in neither blood nor tissue . The focus of infection in cases of proven chronic q fever was endocarditis for 12 case - patients (27%) and endovascular infection for 26 (59%); 6 (14%) had no clear infection focus . Of the case - patients with probable chronic q fever, suspected foci were cardiac valves in 12 (43%), endovascular lesions in 1 (4%), and another focus (e.g., pregnancy or clinical symptoms of infection such as weight loss, night sweats, and fever) in 15 (54%). Long - term antimicrobial drug treatment was started for 40/44 case - patients (91%) with proven chronic q fever, 18/28 case - patients (64%) with probable chronic q fever, and 5/32 case - patients (15%) with possible chronic q fever . Three patients with proven chronic q fever patients died before diagnosis of chronic q fever; 1 refused therapy . In all, 289 controls who had pcr - proven acute q fever in 2009 were sent a questionnaire . Of these, 201 (69.6%) responded, signed the informed consent form, and fulfilled the inclusion criteria (figure 1). Enrollment, selection, and inclusion criteria forcase - patients and controls for case control study to identify risk factors for chronic q fever, the netherlands . Results of the univariate analysis are listed in table 2 . Comparisons for age, vascular history, vascular prosthesis, aneurysm, other vascular surgery, cerebrovascular disease, peripheral vascular disease, valvulopathy, valvular surgery, valvular deviation, ischemic heart disease, other cardiovascular diseases, hypertension, dyslipidemia, diabetes, nonhematologic malignancy (defined as several kinds of solid tumors), renal insufficiency, and pregnancy showed significant differences between case - patients and controls . * patients wth information available for that category . Or, odds ratio; ns, nonsurgical; na, not applicable; copd, chronic obstructive pulmonary disease . Case - patients: aortic valve defects, 10 (no bicuspid valves); mitral valve defects, 9 (no prolapse); tricuspid valve defects, 4 . Controls: aortic valve defects, 6 (no bicuspid valves); mitral valve defects, 3 (1 prolapse). * atrial fibrillation, congestive heart failure, pericarditis, bradycardia, ischemic cardiomyopathy, and left ventricular hypertrophy . Prednisone cumulative dose> 750 mg; use of tumor necrosis factor blocker, methotrexate, mycofenolate mofetil; splenectomy . ##defined as 1014 d of doxycycline treatment results of the multivariate analyses are shown in table 3 . Valvular surgery (or 31.5, 95% ci 3.99249), vascular prosthesis (or 10.4, 95% ci 2.1750.0), aneurysm (or 8.65, 95% ci 1.7442.9), nonhematologic malignancy (or 3.90, 95% ci 1.3311.5), and age (or 1.03, 95% ci 1.011.06) were independently associated with the development of chronic q fever . The final discriminative performance was good, with a c - statistic of 0.71 (95% ci 0.710.83) (figure 2). * or, possible risk factors entered in all analyses: age, vascular prosthesis, aortic aneurysm, other vascular surgeries, peripheral arterial disease, cerebrovascular disease, valvular surgery, valvular disease (nonsurgical), ischemic cardiac disease, other cardiac history, hypertension, dyslipidemia, diabetes, nonhematologic malignancy, renal insufficiency . Immune disorder was also entered in the analyses of proven and probable chronic q fever and of proven chronic q fever . Valvular surgeries in the proven group are subdivided into biological valve (n = 6), prosthetic valve (n = 3), and valve repair (n = 1) all located in the aortic valve (n = 10). Within the controls there were no patients with history of valvular surgery . Locations of vascular prostheses in proven group were infrarenal and iliac (n = 6), infrarenal (n = 4), thoracic (n = 2), and unknown (n = 2). Types of vascular prosthesis were y - prosthesis (n = 7), endovascular aneurysm repair (n = 2), stent graft (n = 2), bentall (n = 1), and unknown (n = 2). Locations of aneurysms in proven group were infrarenal (n = 6), infrarenal and iliac (n = 2), and suprarenal, infrarenal, and iliac (n = 1). Within the control group, #observed stages of chronic kidney disease according to the kidney disease outcome quality initiative guidelines (28) in the proven group were stage 3 (n = 6), stage 4 (n = 2), and stage 5 (n = 1) and in the controls solely stage 3 (n = 2). Goodness - of - fit models for case control study to identify risk factors for chronic q fever, the netherlands . A) all chronic q fever cases (n = 105); area under the curve (c - statistic) 0.77 (95% ci 0.710.83); p<0.001 . B) proven and probable chronic q fever cases (n = 72); c - statistic 0.86 (95% ci 0.810.92); p<0.001 . C) proven chronic q fever cases (n = 44); c - statistic 0.91 (95% ci 0.850.97); p<0.001 . Patient risk factors included in the model (no . Observations): a) valvular surgery (18); vascular prosthesis (15); aneurysm (12); nonhematologic malignancy (16); age, continuous, mean 63.9 y; b) valvular surgery (18); vascular prosthesis (15); aneurysm (12); renal insufficiency (12); age, continuous, mean 67.3 y; c) valvular surgery (10); vascular prosthesis (14); aneurysm (9); renal insufficiency (9); age, continuous, mean 68.4 . Patient risk factors identified in the analysis of the proven cases, representing the most definite chronic q fever cases, were valvular surgery (or 43.6, 95% ci 4.70405), vascular prosthesis (or 26.8, 95% ci 4.88147), aneurysm (or 25.9, 95% ci 4.55147), renal insufficiency (or 16.0, 95% ci 2.06123), and age (or 1.06, 95% ci 1.021.11). The final discriminative performance was good, with a c - statistic of 0.91 (95% ci 0.850.97) (figure 2). To our knowledge, this is the first study that analyzed a large number of potential risk factors for chronic q fever in a large number of patients . Most former studies have been limited by a low number of cases and evaluation of few risk factors . Moreover, quantification of these risk factors was lacking (69,11,12). In our study, we focused mainly on case - patients with proven chronic q fever becausethis group included patients with the most definite form of chronic q fever . Proven chronic q fever also showed the strongest correlation with the identified risk factors . In multivariate analysis, valvular surgery, vascular prosthesis, aneurysms, renal insufficiency, and age were significant risk factors for the development of chronic q fever in patients with proven cases . In the analysis of all patients with chronic q fever cases, nonhematologic malignancy also seemed to be a risk factor; however, this could not be reproduced in the subanalyses of the more definite cases (e.g., proven and probable cases). Valvular surgery, vascular prostheses, and aneurysms were the strongest predictors in this study, which confirms observational findings from earlier studies . A novel finding is the association between mild renal insufficiency and chronic q fever . The majority of patients with chronic q fever and renal disease in our study had stage 3 renal insufficiency according to kidney disease outcome quality initiative guidelines (28). Although terminal renal insufficiency can decrease the immune response, this association was not found for mild renal disease (29). Renal insufficiency is associated with vascular disease, which may explain the elevated incidence of chronic q fever in these patients (30). Increasing age also predisposes for the development of chronic q fever; this predisposition was also illustrated in a recent report of van der hoek et al . The explanation probably lies in the increased prevalence of cardiovascular diseases and the decreased cellular immunity during aging (31,32). Age> 60 years appeared the best cutoff above which the risk for chronic q fever increases significantly . Preexisting cardiac valvulopathy has been found to give an estimated risk of 39% for the development of chronic q fever after infection with c. burnetii (6,33,34). In contrast, recent reports showed no elevated risk for patients with mild valvulopathy in the ongoing outbreak in the netherlands (26,27). Although our univariate analyses showed that nonsurgical cardiac valvulopathy increased the risk for the development of chronic q fever, this finding was not confirmed in the multivariate analysis . This finding can be explained by the fact that 9/17 (53%) case - patients with nonsurgical valvulopathy also had a history of valvular surgery of one of the other valves . The location and type of valvular defects did not differ significantly between case - patients and controls (table 2). A possible explanation for the discrepancy with previous observations lies in the fact that our study was conducted 4 years after start of the q fever epidemic, but chronic q fever endocarditis in patients with nonsurgical cardiac valvulopathy might become evident later (6,8). Furthermore, strain - specific differences in clinical signs and symptoms might also be of importance (26). Presence of valvulopathy in case - patients and controls could have been missed becausethis was assessed only through review of medical records . However, echocardiography, which was standard care for all patients with suspected cases of chronic q fever, revealed no additional congenital or bicuspid valve defects, in comparison to assessment of valvulopathy through review of medical records . From other than the above - mentioned defects, it could not be determined by these echocardiograms if these were preexisting or caused by chronic q fever . Immunosuppression, although not well defined, has been indicated as a risk factor in former reports, but clear definition and statistical empowerment is lacking (8). Although our univariate analysis did show an elevated risk for immunosuppression, especially for patients with proven chronic q fever cases, this elevated risk was not confirmed in multivariate analysis . Immunocompromised patients may be underrepresented in our study becauseit was conducted in a peripheral hospital setting . Pregnancy, another formerly reported risk factor, showed an association with the development of chronic q fever in univariate analysis . However, because there were no pregnant women in the control group and only 3 pregnant women in all case groups, evaluation of pregnancy in multivariate analyses was not possible . A study specifically designed to evaluate associations between pregnancy and q fever is ongoing in the netherlands (35). In our opinion, our data were representative for this large q fever outbreak because they were well documented data and patients were willing to participate . The fact that case - patients and controls were living in the same area increases the comparability of these groups and strengthens the results . First, all controls had an acute episode in 2009, but information about their signs and symptoms was obtained in 2011, introducing possible recall bias . We tried to reduce this bias by requesting additional information from the general practitioners and by reviewing clinical test results and physicians reports in the hospital registration systems . Because information bias could also have been introduced by the subjective interpretation of physicians reports for case - patients and controls, 2 of our researchers interpreted the results independently . Serologic follow - up of the controls after the acute q fever episode lasted only 1 year, which is the normal follow - up period in the netherlands . However, because chronic q fever can become manifest years after initial infection, development of chronic q fever after this follow - up period is still possible (6,22). Still, 75% of chronic q fever cases develop within 6 months after primary infection (22). Moreover, according to the observed decrease in antibody titers of these patients, progression to chronic q fever is not likely . In addition, as a consequence of the inclusion of patients with symptomatic acute q fever as a control group, the results can only be generalized to patients with symptomatic acute q fever, although the results probably provide an adequate indication of risks factors for patients with mild or asymptomatic primary q fever . Notably, almost all controls received antimicrobial drug treatment at time of acute q fever, in contrast to the case - patients, among which only a minority had symptomatic acute q fever . Thus, antimicrobial drug treatment might influence the chance of chronic q fever development, although there is no quantitative evidence that treatment for acute q fever reduces the chance for chronic q fever (4). Chronic q fever cases were selected and classified according to the definitions of the dutch q fever consensus group (20), which still need confirmation . The definition of probable chronic q fever contains several patient criteria that we also included as potential risk factors in our study (e.g., valvular disease, vascular prosthesis, aneurysm, and immunosuppressive state). Nevertheless, proven chronic q fever, for which these criteria are not part of the definition, was also predicted with the identified risk factors in multivariate analysis, thereby confirming the independent risk association of these variables . Some chronic q fever cases were identified during screening programs for patients who had valvular surgery, aneurysms, or vascular prostheses . Patients with these risk factors may therefore be overrepresented within our study, although all proven case - patients had symptomatic disease . Last, the results of this study have to be considered in view of a predominant c. burnetii strain that is responsible for the majority of q fever cases in humans in the netherlands (36). Worldwide, q fever manifestations differ geographically, which might result from differences in c. burnetii strains (4). In conclusion, previous valvular surgery, vascular prosthesis, aneurysms, renal insufficiency, and age were identified as major risk factors for the development of chronic q fever among persons infected with c. burnetii . Because untreated chronic q fever comes with serious risk for illness and death, awareness is required in people with acute q fever possessing the identified risk factors . This may require close follow - up or even prophylactic treatment in high - risk groups . Moreover, in case of large q fever outbreaks, screening is advisable for patients with these identified risk factors.
They are usually encountered as incidental findings in magnetic resonance imaging (mri), or in arthroscopy . They may originate from both the cruciate ligaments and the menisci, from the popliteus tendon and alar folds, infrapatellar fat pad of hoffa, and subchondral bone cysts . Those arising from the hoffa s fat pad, usually present as palpable mass at anterior aspect of the knee joint . We report a case of intraarticular ganglion cyst of knee arising from the infrapatellar fat pad and protruding anterolaterally through retinacular rent into the subcutaneous plane . A 19-year - old young man, presented with a painless gradually increasing swelling at the anterior aspect of left knee of 9 months duration . Mri scan revealed a multilobulated, cyst with septations within the anterior aspect of the knee joint, just inferolateral to the patella, with deep extension into the infrapatellar fat pad, and superficial extension into the subcutaneous space across the retinaculum . After diagnostic arthroscopy, we performed an open excision of the cystic mass and confirmed the retinacular rent pre - operatively . Arthroscopic resection and debridement is the gold standard treatment in ganglion cyst of the knee . However, a subcutaneous extension may lead to incomplete arthroscopic resection: leaving behind the residual tissue which may cause recurrence . Therefore, proper pre - operative evaluation of mr images of these cases is very important . A ganglion is a cystic tumor - like lesion of unknown etiology, surrounded by a dense network of connective tissue, filled with a gelatinous fluid containing hyaluronic acid and other mucopolysaccharides . Intra - articular ganglion cysts of the knee joint are uncommon . They are usually encountered as incidental findings in magnetic resonance imaging (mri), or in arthroscopic examination . They may originate from both the cruciate ligaments and the menisci, from the popliteus tendon and alar folds, infrapatellar fat pad of hoffa, and subchondral bone cysts . Those arising from the hoffa s fat pad have been reported to present as palpable mass at anterior aspect of knee joint [2 - 5]; however, to the best of our knowledge, none of them had subcutaneous communications across the retinaculum . We report a case of intra - articular ganglion cyst of knee arising from the infrapatellar fat pad and protruding anterolaterally through retinacular rent into the subcutaneous plane . A.a.m ., a 19-year - old young man, presented with a painless gradually increasing swelling at the anterior aspect of left knee for last 9 months . There was no history of trauma, local injections, instability, locking episodes, clicking, snapping, or any constitutional symptoms . Clinical examination revealed a 2.5 cm x 2.5 cm round, soft, cystic, non - tender, irreducible swelling at anterolateral aspect of left knee 1.5 cm above the lateral joint line just lateral to the patellar tendon with discoloration of the overlying skin . It was partially mobile, not fixed to the extensor mechanism or overlying skin, and was more prominent in knee extension (fig . 1). He could squat and sit cross - legged without any discomfort with a normal range of movement . There was no effusion, no joint line tenderness, and no evidence of ligamentous laxity . Pre - operative clinical pictures showing the ganglion cyst at anterolateral aspect of left knee in various degrees of knee flexion . Mri scan revealed a multilobulated, cyst with septations within the anterior aspect of the knee joint, just inferolateral to the patella, with deep extension into the infrapatellar fat pad . (a) normal radiograph except increased soft tissue shadow, (b) parasagittal and axial mr images showing the ganglion cyst from infrapatellar fat pad extending subcutaneously across the retinaculum . Then we went for open excision of the cystic mass through lateral parapatellar approach (incision included that of the anterolateral arthroscopic portal). The cyst was found to be protruding just beneath the skin through a retinacular rent (fig . However, the entire mass was taken out, but we had to sacrifice the fat pad almost entirely . Post - operatively the patient underwent appropriate rehabilitation making an uneventful recovery and returned to his normal activities . He was symptom - free in his last follow - up at 6 months post - operatively . Between the first description of an anterior cruciate ligament (acl) ganglion by caan in 1924 during a routine autopsy and the early 1990s, only a handful of sporadic cases were described in the literature . The last 30 years have seen a huge increase in the number of these cases being reported owing to the widespread use of both mri and arthroscopy . The prevalence of intra - articular cystic knee masses is 1.3% in mri and 0.6% in arthroscopy . Several theories have been suggested: (a) synovial tissue herniation, (b) mucinous degeneration of connective tissue after trauma, (c) synovial ectopia or displacement during embryogenesis, etc . . The histological finding that ganglia are fluid - filled structures without any epithelial lining confirms that they are not true cysts, and thus favors the degenerative theory over others; and, even though, ganglia may develop in the absence of trauma, it is postulated that repetitive microtrauma from joint and soft tissue motion may play an important role . Intra - articular ganglia of knee originates most commonly from the acl accounting for more than 50% cases; whereas, the location at infrapatellar fat pad is far less common (approximately 4%). However, only 10% of them are symptomatic with most being incidental finding on mri or arthroscopy . The symptoms are usually non - specific, like knee pain, locking, clicking or popping sensation, decreased range of motion, etc ., and depend on the site and size of the cyst . Ganglia anterior to the acl tend to limit extension, whereas those posterior to the posterior cruciate ligament often limit knee flexion . Mri is the investigation of choice because it is the most sensitive, specific accurate, and noninvasive method for depicting such cystic masses, including their size and location, and to detect associated intra - articular pathologies . For intra - articular ganglion cyst in the infrapatellar fat pad, fat - suppressed contrast - enhanced mri could be useful, because a thin, rim - enhancing feature of intra - articular ganglion cyst allows it to differentiate from synovial hemangioma and synovial sarcoma . Among the various treatments available, arthroscopic resection and debridement are currently the preferred procedure; open surgery may be necessary in particular cases only . After diagnostic arthroscopy, we performed an open excision of the ganglion cyst since it was not clearly visible on arthroscopy; moreover, we did not have any previous experience of arthroscopic resection of intra - articular ganglion cysts . Hence, in view of the high recurrence rate after incomplete removal of ganglion, we preferred open excision over arthroscopic resection . Although recurrence is rare after arthroscopic or open surgical treatment of ganglion cyst of knee, the fact that it does occur in other areas even after excision, this patient should be followed up on regular basis . Current literature suggests arthroscopic resection and debridement as the gold standard treatment of ganglion cyst of the knee . However, a subcutaneous extension may lead to incomplete arthroscopic resection: leaving behind the residual tissue which may cause recurrence . Therefore, proper pre - operative evaluation of mr images of these cases is very important . They are usually encountered as incidental findings in mri, or in arthroscopy . Arthroscopic resection and debridement is regarded as the gold standard treatment in ganglion cyst of the knee . However, very rarely, these masses may extend subcutaneously across the retinaculum . This may lead to incomplete arthroscopic resection: leaving behind the residual tissue which may cause recurrences . Therefore, proper pre - operative evaluation of mr images of these cases is very important.
The caudate lobe lies deep in the liver, between the hepatic hila and the retrohepatic inferior vena cava (ivc), and is adjacent to the major hepatic veins in its upper part . Although the caudate lobe constitutes only a small part of the whole liver, it has the same histologic structure and the same incidence of developing benign and malignant neoplasms as other hepatic segments in proportion to their volume . Percutaneous ethanol injection and radiofrequency ablation (rfa) for tumors in the caudate lobe are difficult to be carried out because of their spatial peculiarity . Multiple bilateral blood supplies from hepatic artery and portal vein make transcatheter arterial chemoembolization (tace) less effective for malignant tumors in the lobe than those in the main lobes . Surgical resection is left the only radical solution for symptomatic benign tumors and malignant tumors confined to the lobe . Isolated caudate lobectomy, a parenchyma - sparing procedure, is still a challenge for hepatobiliary surgeons, especially in cirrhotic patients . 16 cases of isolated caudate lobectomy in our departments from january 2010 to december 2013 were reviewed to optimize the operation . According to kumon's nomenclature, the caudate lobe consists of 3 portions: the spiegel lobe (i.e., couinaud's segment i), the paracaval portion (i.e., couinaud's segment ix), and the caudate process . The spiegel lobe locates behind the lesser omentum, to the left of the retrohepatic ivc . The paracaval portion, which is attached to the anterior surface of the retrohepatic ivc by the retrohepatic ligament and the short hepatic veins, lies to the right of the spiegel lobe . The caudate process, the smallest part of the three, is a thin tongue - like projection between the ivc and the portal vein . Isolated caudate lobectomy is to remove either part or total of the lobe surgically (i.e., isolated partial or complete caudate lobectomy). Classified hepatocellular carcinomas spread from the caudate lobe into five types, which were frequently adopted to describe all neoplasms that originated in the caudate lobe . They were as follows: type 1 lesions: lesions in the upper part of the spiegel lobe;type 2 lesions: lesions in the lower part of the spiegel lobe;type 3 lesions: lesions in the paracaval portion;type 4 lesions: lesions in the caudate process;type 5 lesions: lesions spread from the whole caudate lobe . Type 1 lesions: lesions in the upper part of the spiegel lobe; type 2 lesions: lesions in the lower part of the spiegel lobe; type 3 lesions: lesions in the paracaval portion; type 4 lesions: lesions in the caudate process; type 5 lesions: lesions spread from the whole caudate lobe . 16 cases of isolated caudate lobectomy were performed for neoplasms confined to the caudate lobe, including seven cases of hepatocellular carcinoma (7/16, 43.75%), four cases of hepatic cavernous hemangioma (4/16, 25%), one case of hepatocellular adenoma (1/16, 6.25%), one case of inflammatory pseudotumor (1/16, 6.25%), one case of hepatic hamartoma (1/16, 6.25%), one case of mixed hepatocellular carcinoma and cholangiocellular carcinoma (1/16, 6.25%), and one case of metastatic colonic cancer (1/16, 6.25%). Hepatitis b virus surface antigen was positive in all the seven cases of hepatocellular carcinoma and in the case of mixed hepatocellular carcinoma and cholangiocellular carcinoma, respectively, and hepatitis b virus surface antibody was positive in the case of hepatocellular adenoma . The tumors were measured in the maximum diameter from 2 cm to 12 cm (4.91 cm in average). According to hasegawa et al . 's classification, there were one case of type 1 lesions, three cases of type 2 lesions, three cases of type 3 lesions, six cases of type 4 lesions, and three cases of type 5 lesions (table 1). Isolated resection of the caudate lobe consisted of four major steps: mobilization of the lobe, outflow control by dividing the short hepatic veins behind the lobe, inflow control by dividing the portal triads to the lobe, and division of the hepatic parenchyma between the caudate lobe and the main liver . Left side approach, or right side approach, or both left and right sides approach were adopted in the operation . The sequence of the four steps and the surgical approach alternated according to the tumor's location, size, texture, and nature . Was adopted for resection of small masses that originated in the spiegel lobe, especially type 2 lesions . After entering the abdominal cavity through a reversed l - shaped incision on the right upper quadrant of the belly, the round, falciform, left triangular, left coronary and hepatogastric ligaments the retroperitoneum covering the left wall of the retrohepatic ivc was incised to free the left margin of the spiegel lobe . Then, the spiegel lobe was easily elevated from the retrohepatic ivc to expose the short hepatic veins to direct view as they were divided and ligated caudal cranially . The left portal hilum was lifted ventrally to expose the portal triads to the spiegel lobe, which were divided and ligated subsequently . Right side approach was adopted for resection of small masses that originated in the caudate process . After entering the abdominal cavity through a reversed l - shaped incision, the round, falciform, right triangular, right coronary and hepatorenal ligaments were divided in turn . The liver was elevated from the retrohepatic ivc and rotated to the left to show the short hepatic veins, which were divided and ligated in a cranial direction . The possible inferior right hepatic vein (irhv), which often enters the ivc near the right adrenal gland vein, the retrohepatic ligament was divided when necessary from the left lateral surface of the ivc through an incision on the lesser omentum . If the portal triads to the caudate process could be dissected easily, they would be divided in advance; in reverse, they were controlled during dividing the liver parenchyma by cusa . Left and right side approach was adopted for resection of the majority of caudate masses . A reversed l - shaped incision on the right upper quadrant was adequate in most both sides approach cases . The transverse arm of the incision might be extended to the left subcostal region if necessary . The falciform ligament was dissected to the ventral surface of the suprahepatic ivc to show the loose space between the right hepatic vein and the confluence of the left and middle hepatic veins . The retroperitoneum covering the infrahepatic ivc and the left wall of the retrohepatic ivc (referred to as makuuchi's fascia in japanese literature) was incised caudal cranially to the superior recess of the lesser sac to expose the entire retrohepatic ivc . At this time the confluence of the left and middle hepatic veins, which enters the anterior left lateral wall of the suprahepatic ivc, was encircled by passing a vascular tape through the latent space surrounded by the dorsal surface of the two hepatic veins, the ventral surface of the ivc, and the tip of the caudate lobe . The liver was lifted and all the short hepatic veins (including the irhv) were taken down and suture - ligated . The hepatoduodenal ligament was loosened, and the duodenum and the pancreatic head were partly mobilized by kocher maneuver for better exposure of large caudate masses . After the liver parenchyma between the caudate lobe and the main liver was transected, the tumors were removed en bloc . Division of the ligament enabled the elevation of the caudate lobe from the caval vein . But when an enlarged spiegel lobe embraced the ivc dorsally, it should be divided from the right side more conveniently . The venous ligament (i.e., the arantius' ligament) lies in the sulcus of the ligamentum venosum and connects the left portal vein to the root of the left hepatic vein . The venous ligament was divided near the left hepatic vein to partly release the tip of the caudate lobe and to facilitate the isolation of the confluence of the left and middle hepatic veins . The hepatic pedicle was encircled with a vascular tape for possible temporary inflow control of the liver . After complete mobilization of the liver and incision of the retroperitoneum that covers the infrahepatic ivc and the left wall of the retrohepatic ivc, the suprahepatic and infrahepatic ivc were isolated and encircled with vascular tapes easily in case of possible temporary total hepatic blood occlusion . These veins were best approached from the right side when there was a huge tumor . The portal hila were lifted to expose the portal triads to the caudate lobe, which originate mainly from the left hilum and secondarily from the bifurcation (figure 2). The parenchymal bridge between the caudate lobe and segments iv, viii, and vii was transected by cusa . Some small inflow vasculatures and draining vessels to the main hepatic veins, which were difficult to be dissected and ligated beforehand, were divided during transecting the liver parenchyma . All 16 cases of isolated caudate lobectomy were accomplished successfully without death and severe complications . Left side approach was adopted in two cases (2/16, 12.5%) and right side approach in three cases (3/16, 18.75%), while both sides approach in 11 cases (11/16, 68.75%). Estimated intraoperative blood loss ranged from 100 ml to 850 ml (356.25 ml in average) and transfusion varied from 0 to 800 ml (137.5 ml in average). Pringle maneuver was adopted in six cases for temporary inflow control of the liver (occlusion time ranged from 6 min to 13 min). The confluence of the left and middle hepatic veins (and the right hepatic vein in two cases) was taped in five cases for regional outflow control . The suprahepatic and infrahepatic ivc were encircled with vascular tapes in two cases, but neither needed total hepatic blood occlusion of the hepatic hila and the ivc at the same time . Small leakage in the retrohepatic ivc or the major hepatic veins was encountered in five cases, which was repaired with prolene suture . Total operative time ranged from 150 min to 270 min (211.25 min in average). No bile leakage was encountered, which was reported as the major complication after isolated caudate lobectomy . The seven cases of hepatocellular carcinoma and the case of mixed hepatocellular carcinoma and cholangiocellular carcinoma were followed up from 6 to 28 months, with one death case due to liver failure and upper gastrointestinal hemorrhage in the 26th month after operation and two cases of recurred hepatocellular carcinoma in the main liver, who thereafter received tace, percutaneous rfa, and systemic chemotherapy . The regular hepatectomy techniques are seldom employed in the operation . The surgical strategy chosen for each patient must be based on the patient's idiographic situation . In the 16 cases of isolated caudate lobectomy, left side approach was adopted in two cases, right side approach in three cases, and combined left and right sides approach in 11 cases . Left side approach is adopted for small lesions in the lower part of the spiegel lobe and right side approach for small lesions in the caudate process . Combined left and right sides approach is recommended for the majority of neoplasms in the caudate lobe [5, 9], especially for those that are bigger than 4 cm in diameter, those originating in the paracaval portion or in the whole caudate lobe, or those that are thought to be malignant tumors, which require total caudate lobectomy for clearance of the tumors . Symptomatic and continuously enlarging hepatic cavernous hemangioma also requires total caudate lobectomy before it reaches a nonresectable size . In fact, complete caudate lobectomy is technically easier and controllable than partial resection of the lobe . Loosening the hepatoduodenal ligament and performing the kocher maneuver help to expose the caudate lobe better and provide more space for the operation . When a bulky caudate tumor protrudes into the space between the right and middle hepatic veins or compresses the major hepatic veins severely, the anterior transhepatic approach should be employed in addition [10, 11] or combined lobectomy should be adopted . Thorough medical imaging study of ct or / and mri scan and type - b ultrasonography before operation is mandatory to illuminate the anatomic relationship between the masses and the hepatic hila, the major hepatic veins, and the retrohepatic ivc and to rule out metastases of malignant tumors . Although thrombosis in the portal vein is not thought to be a contraindication for isolated caudate lobectomy, ruling out thrombus in the portal vein and the ivc is important for the choice of strategy of treatment . Usually it is easy to dissect and divide the short hepatic veins along a tumor - free plane on the ventral surface of the retrohepatic ivc unless the tumor has involved the caval wall substantially . Unlike the high incidence of thrombosis in the portal vein or the retrohepatic ivc in advanced hepatocellular carcinoma in the main liver or hilar cholangiocarcinoma which have involved the caudate lobe, the incidence of thrombosis is much lower than that expected for caudate tumors . Dissection and division of the short hepatic veins exposure of the entire course of the retrohepatic ivc by incising the covered retroperitoneum is of utmost importance . The superior recess of the lesser sac extends rightwards behind the spiegel lobe and the retrohepatic ivc . The short hepatic veins are first divided before the portal triads to separate the caudate lobe from the ivc, in case of removing the lobe quickly after temporary hemostasis of a possible huge bleeding from a parenchymal laceration or a tear in the major hepatic veins by pringle maneuver . Large short hepatic veins should be tied and sutured on the hepatic side . On the caval side, the large dissected end should be repaired with prolene suture after applying a side - wall vascular clamp . The largest short hepatic vein drains the bulkiest part of the caudate lobe, which locates predominantly in the middle of the spiegel lobe and secondarily around the central part of the paracaval portion . . A medium - sized short hepatic vein that drains the tip of the caudate lobe to the left hepatic vein or the nearby ivc is sometimes found, which should be ligated with care so as not to be torn . A broken hepatic vein is repaired more conveniently after the caudate lobe is moved out as it is facilitated by pringle maneuver . If combined with occlusion of the confluence of the left and middle hepatic veins (and sometimes the right hepatic vein), a more clear operative field is presented with less possibility of air embolism . In contrast, the caudate lobe attaches to the ivc circumferently by retrohepatic ligament and adheres to the caudal side of left liver by the venous ligament . When the caudate lobe is small or when the tumor locates mainly in the caudate process, the retrohepatic ligament is divided from the left side easily . But when the caudate lobe is enlarged, especially when it embraces the retrohepatic ivc dorsally with a bulky spiegel lobe, the retrohepatic ligament should be divided from the right side . Division of the retrohepatic ligament makes it much easier to elevate the caudate lobe from the ventral surface of the retrohepatic ivc, facilitates the exposure and division of the short hepatic veins, and contributes to the isolation of the main hepatic veins likewise . The irhv should be divided beforehand, which will facilitate the management of the other short hepatic veins from the right side . The division of the venous ligament loosens the cranial polar of the caudate lobe, facilitates mobilizing the caudate lobe from the left side, provides more space for encircling the left and middle hepatic veins, and minimizes the possibility of injuring the veins . Hepatocellular carcinoma usually occurs after hepatitis b in china, no matter where the first tumor appears . After the original hepatic cancer in the caudate lobe is resected, the tumor may easily recur in the main liver lobes . Close follow - up of these patients at an interval of one to two months is necessary . Chemotherapy, transarterial embolization or chemoembolization, percutaneous rfa, and alcohol injection are adopted to reduce the possibility of recurrence or to treat a recurred tumor . Many authors have reported comparable (or even better) survival rate after isolated resection of caudate hepatocellular carcinoma with those in the main liver [1, 14, 15], which suggests that isolated caudate lobectomy for hepatocellular carcinoma in the caudate lobe is practical . In summary, left and right side approach is adopted for isolated resection of most neoplasms in the caudate lobe . Mobilizing the whole caudate lobe is more important than enucleating the tumor itself in the operation . Considering the patients' safety as well as eradication of the diseases, the best surgical strategy is the one that is uncomplicated and easy to be mastered by most hepatobiliary surgeons, with fewer traumas and complications . And under the principle of mobilizing the caudate lobe firstly, controlling the outflow and then inflow vessels secondly, and dividing the liver parenchyma thirdly, it is advised that the relatively easier and safer step should be carried out first . For example, some short hepatic veins can be divided while dividing the retrohepatic ligament; and some portal triads to the caudate lobe can be divided while dividing the liver parenchyma . Patients with malignant tumors should be followed up regularly after operation for recurrence and for adjuvant therapy.
This investigation is based on cross - sectional data from 275 independently living individuals who participated in the follow - up in 2008 of the longitudinal study on nutrition and health status of senior citizens of giessen (gisela study), germany (50.6north). Investigations took place in the institute of nutritional science in giessen from july to october . The ethical committee of the faculty of medicine at the justus - liebig - university, giessen, approved the research protocol . Exclusion criteria for the present study were any of the following conditions: incomplete data (n=23) on dietary assessment and biochemical parameters, including serum 25-hydroxyvitamin d3 [25(oh)d3] and parathyroid hormone; chronic renal disease (n=5); and lifetime history of cancer of the gastrointestinal tract (stomach, small intestine, colon; n=11). Fasting blood samples were collected, and serum aliquots were stored at 70c until further analysis . Serum 25(oh)d3 and parathyroid hormone were measured by an electrochemiluminescence immunoassay (roche diagnostics gmbh, mannheim, germany). Participants completed a self - administered questionnaire on socioeconomic and lifestyle characteristics, such as age, sex, monthly household net income, smoking status, physical activity, and disease history . Bmi was defined as weight (kg) divided by height squared (m). Percentage total body fat (% tbf) was determined by a single - frequency (50 khz) bioelectrical impedance analyser (akern - rjl bia 101/s; data input, frankfurt, germany), according to manufacturer's instructions and the predictive formula of roubenoff et al . Nutritional intake was determined using a 3-day estimated dietary record, which was developed and validated for the gisela study (20). Validity was evaluated by nitrogen excretion in the 24-h urine in relation to protein intake, and by comparing energy intake and resting metabolic rate assessed by indirect calorimetry according to goldberg et al . For every food item, both typical household measures (e.g. Slice, cup and spoon) and the appropriate weights were given . The participants were instructed to record their entire food intake on three consecutive days directly after consumption, starting on a sunday . Food, energy, and nutrient intakes were calculated using the german food code and nutrient data base (federal institute for health protection of consumers and veterinary medicine, berlin, germany) (22) version ii.3 . For the present analysis, we created nine food groups to measure the contribution of particular food categories to vitamin d intake: fish / fish products (e.g. Fish, canned fish, seafood); eggs; fats / oils (e.g. Butter, lard, margarine, oil); bread / bakery products (e.g. Bread, toast, cake, biscuits); milk / dairy products (e.g. Milk, cheese, yoghurt, curd, cream); potatoes / fruits/ vegetables and related products; nutriments (e.g. Pasta, rice, cereals, corn flakes); meat / meat products (e.g. Meat, innards, sausages, cold cuts, ham); and the category others inclusive of, for example, sauces, sweets, snacks, and beverages . The contribution of vitamin d by eggs and fats via processed foods was captured by the foods to which these components have been added during processing or, in the case of fats, of which they are an inherent component . In addition, subjects were asked to rank their usual fish, egg, and milk consumption frequencies on a scale from never to daily intake . Continuous data are expressed as mean and standard deviation (sd) and median and 5th to 95th percentile, when appropriate . Chi - square test or fisher's exact test was used to assess differences in proportions . We performed stratified analyses by sex (females vs. males), median age (<76 vs. 76 years), median vitamin d status (63 vs.> 63 nmol / l), median bmi (26.8 vs.> 26.8 kg / m), and household net income (<1,500 vs. 1,500 /month) to examine whether vitamin d intake and the contributing food groups differed between the respective groups . Spearman correlation was applied to evaluate correlations between vitamin d intake and sex, age, bmi,% tbf, pal, smoking behaviour (never smokers vs. current and ex - smokers), household net income (<1,500 vs. 1,500 /month), nutritional parameters, and 25(oh)d3 . Those variables that showed a significant association with vitamin d intake were entered in the multiple backward regression model with the logarithmically transformed (log) vitamin d intake as dependent variable . Statistical analyses were conducted with spss 21.0 for windows (ibm, chicago, usa). This investigation is based on cross - sectional data from 275 independently living individuals who participated in the follow - up in 2008 of the longitudinal study on nutrition and health status of senior citizens of giessen (gisela study), germany (50.6north). Investigations took place in the institute of nutritional science in giessen from july to october . The ethical committee of the faculty of medicine at the justus - liebig - university, giessen, approved the research protocol . Exclusion criteria for the present study were any of the following conditions: incomplete data (n=23) on dietary assessment and biochemical parameters, including serum 25-hydroxyvitamin d3 [25(oh)d3] and parathyroid hormone; chronic renal disease (n=5); and lifetime history of cancer of the gastrointestinal tract (stomach, small intestine, colon; n=11). Fasting blood samples were collected, and serum aliquots were stored at 70c until further analysis . Serum 25(oh)d3 and parathyroid hormone were measured by an electrochemiluminescence immunoassay (roche diagnostics gmbh, mannheim, germany). Participants completed a self - administered questionnaire on socioeconomic and lifestyle characteristics, such as age, sex, monthly household net income, smoking status, physical activity, and disease history . Bmi was defined as weight (kg) divided by height squared (m). Percentage total body fat (% tbf) was determined by a single - frequency (50 khz) bioelectrical impedance analyser (akern - rjl bia 101/s; data input, frankfurt, germany), according to manufacturer's instructions and the predictive formula of roubenoff et al . Nutritional intake was determined using a 3-day estimated dietary record, which was developed and validated for the gisela study (20). Validity was evaluated by nitrogen excretion in the 24-h urine in relation to protein intake, and by comparing energy intake and resting metabolic rate assessed by indirect calorimetry according to goldberg et al . For every food item, both typical household measures (e.g. Slice, cup and spoon) and the appropriate weights were given . The participants were instructed to record their entire food intake on three consecutive days directly after consumption, starting on a sunday . Food, energy, and nutrient intakes were calculated using the german food code and nutrient data base (federal institute for health protection of consumers and veterinary medicine, berlin, germany) (22) version ii.3 . For the present analysis, we created nine food groups to measure the contribution of particular food categories to vitamin d intake: fish / fish products (e.g. Fish, canned fish, seafood); eggs; fats / oils (e.g. Butter, lard, margarine, oil); bread / bakery products (e.g. Bread, toast, cake, biscuits); milk / dairy products (e.g. Milk, cheese, yoghurt, curd, cream); potatoes / fruits/ vegetables and related products; nutriments (e.g. Pasta, rice, cereals, corn flakes); meat / meat products (e.g. Meat, innards, sausages, cold cuts, ham); and the category others inclusive of, for example, sauces, sweets, snacks, and beverages . The contribution of vitamin d by eggs and fats via processed foods was captured by the foods to which these components have been added during processing or, in the case of fats, of which they are an inherent component . In addition, subjects were asked to rank their usual fish, egg, and milk consumption frequencies on a scale from never to daily intake . Continuous data are expressed as mean and standard deviation (sd) and median and 5th to 95th percentile, when appropriate . Chi - square test or fisher's exact test was used to assess differences in proportions . We performed stratified analyses by sex (females vs. males), median age (<76 vs. 76 years), median vitamin d status (63 vs.> 63 nmol / l), median bmi (26.8 vs.> 26.8 kg / m), and household net income (<1,500 vs. 1,500 /month) to examine whether vitamin d intake and the contributing food groups differed between the respective groups . Spearman correlation was applied to evaluate correlations between vitamin d intake and sex, age, bmi,% tbf, pal, smoking behaviour (never smokers vs. current and ex - smokers), household net income (<1,500 vs. 1,500 /month), nutritional parameters, and 25(oh)d3 . Those variables that showed a significant association with vitamin d intake were entered in the multiple backward regression model with the logarithmically transformed (log) vitamin d intake as dependent variable . Statistical analyses were conducted with spss 21.0 for windows (ibm, chicago, usa). However, 25(oh)d3 levels <50 nmol / l were noticed in 21.3% of the subjects, while 21.3% had 25(oh)d3 levels 75 both sexes failed to meet the recommended daily intake of 20 g vitamin d (9); only one woman and two men ingested 20 g / day . Vitamin d intakes <5 g / day and <10 g / day were observed in 57.4 and 90.2% of the study population, respectively . Sex differences were not found in any of these analyses (p>0.05). Of the subjects who made statements on their usual consumption frequencies of fish (n=226), eggs (n=229), and milk (n=212), 2.2% (n=5), 0.9% (n=2), and 24.1% (n=51) were non - consumers of fish, eggs, and milk; 75.2% (n=170), 76.4% (n=175), and 26.4% (n=56) reported to eat seldom or several times per month fish, eggs, and milk; whereas 22.6% (n=51), 22.7% (n=52), and 49.5% (n=105) stated that they consume fish, eggs, and milk several times per week or daily, respectively . Whitney u test, chi - square test, and fisher's exact test for analysing sex differences; missing data were present on total body fat (n=5), physical activity level (n=26), smoking behaviour (n=2), and monthly household net income (n=41). Table 2 presents the median values and the 5th and 95th percentiles of the daily intake levels of the nine food groups, the corresponding absolute vitamin d intake by these food groups, and the relative contributions of the food groups to the daily vitamin d intake . For some of the food groups (eggs, potatoes / fruits / vegetables, nutriments, meat / meat products, and others), median values for their contribution to vitamin d intake amounted to zero this is due to the very low amount of vitamin d in these food groups and/or the fact that within the 3 days of dietary record, more than half of the subjects consumed no such foods (as in the case of eggs, for example). Median and mean percentage contributions of food groups to the daily vitamin d intake differed considerably, indicating non - normally distributed data . When expressed as mean values, contributions to the daily vitamin d intake amounted to approximately 40% for fish / fish products followed by eggs (15%), fats / oils (13%), bread / bakery products (12%), and milk / dairy products (12%). The other food groups altogether added to less than 10% to the mean daily vitamin d intake . Daily intake levels of food groups and corresponding vitamin d intake (n=235) stratifying our analysis by age (<76 vs. 76 years), vitamin d status tended to be higher in younger subjects compared to older subjects (median: 65.2 vs. 61.2 nmol / l; p=0.053), whereas dietary vitamin d intake was significantly higher in older participants (median: 2.4 vs. 4.1 g / day; p=0.049). Apart from a tendency towards a higher contribution of fish to the vitamin d intake in older participants (35 vs. 44%; p=0.102), contributing food groups did not differ between age groups . No substantial differences in vitamin d intake levels or in relative contributions of food groups were found after stratifying the cohort according to the median vitamin d status (p>0.05). However, a slightly higher contribution of meat / meat products to the dietary vitamin d intake in subjects with a vitamin d status 63 nmol / l compared to subjects who had a higher vitamin d status was noticed (2.05 vs. 2.02%; p = 0.024). After stratifying the cohort into two groups based on the median bmi, vitamin d status of subjects with a bmi 26.8 kg / m was higher than in subjects with a bmi>26.8 kg / m (median: 67.3 vs. 59.3 no significant differences in vitamin d intake or in contributions of food groups were found (p>0.05). Similar results were observed when the cohort was divided according to the sex - specific median% tbf (p>0.05). Subsequent to the separation of the study population with regard to the monthly household net income (<1,500 vs. 1,500), vitamin d status was higher in the upper income group (median: 59.4 vs. 65.6 nmol / l; p=0.020), while vitamin d intake was significantly lower (median: 5.5 vs. 2.7 g / day; p=0.007). Compared to subjects reporting an income 1,500 /month, subjects with an income <1,500 /month showed higher mean fractional contributions to dietary vitamin d intake by fish (49 vs. 36%; p=0.028) and by potatoes / fruits / vegetables (2.4 vs. 2.3%, p=0.044), whereas the contributions of fats / oils (9 vs. 16%, p=0.010) and, albeit not significant, of milk / dairy products (9 vs. 13%, p=0.066) were lower . Dietary vitamin d intake was associated with age,% tbf, intake of energy, fat and alcohol, frequency of fish intake, and household net income, but did not correlate with sex, bmi, smoking, pal, use of vitamin d supplements, and consumption frequencies of eggs and milk . Except for a positive correlation with alcohol intake (r s=0.152; p=0.020), 25(oh)d3 levels did not correlate with dietary parameters (p>0.05). Spearman correlations between vitamin d intake and relevant parametersa r s = spearman correlation coefficient . Missing data were present on total body fat (n=5); consumption frequencies of fish (n=9), eggs (n=6), and milk (n=23); physical activity level (n=26); smoking behaviour (n=2); and monthly household net income (n=41). Frequencies were dichotomised in never to several times per month (coded as 0) versus several times per week to daily (coded as 1). Multiple regression analysis (n=185) was performed with log vitamin d intake as the dependent variable and age,% tbf, intake of energy, fat and alcohol, frequency of fish intake, and household net income as independent variables . Thereby, fat intake (=0.283; p<0.0001) and frequency of fish consumption (=0.235; p=0.001) were identified as positive determinants of dietary vitamin d intake, whereas alcohol intake (=0.122; p=0.082), net income (=0.174; p=0.013), and% tbf (=0.174; p=0.014) negatively affected vitamin d intake . This regression model explained 19.6% of the variance in dietary vitamin d intake in elderly subjects . The exclusion of two people with very high dietary vitamin d intake levels (26 and 37 g / day) provided approximately equivalent results . However, 25(oh)d3 levels <50 nmol / l were noticed in 21.3% of the subjects, while 21.3% had 25(oh)d3 levels 75 both sexes failed to meet the recommended daily intake of 20 g vitamin d (9); only one woman and two men ingested 20 g / day . Vitamin d intakes <5 g / day and <10 g / day were observed in 57.4 and 90.2% of the study population, respectively . Sex differences were not found in any of these analyses (p>0.05). Of the subjects who made statements on their usual consumption frequencies of fish (n=226), eggs (n=229), and milk (n=212), 2.2% (n=5), 0.9% (n=2), and 24.1% (n=51) were non - consumers of fish, eggs, and milk; 75.2% (n=170), 76.4% (n=175), and 26.4% (n=56) reported to eat seldom or several times per month fish, eggs, and milk; whereas 22.6% (n=51), 22.7% (n=52), and 49.5% (n=105) stated that they consume fish, eggs, and milk several times per week or daily, respectively . Whitney u test, chi - square test, and fisher's exact test for analysing sex differences; missing data were present on total body fat (n=5), physical activity level (n=26), smoking behaviour (n=2), and monthly household net income (n=41). Table 2 presents the median values and the 5th and 95th percentiles of the daily intake levels of the nine food groups, the corresponding absolute vitamin d intake by these food groups, and the relative contributions of the food groups to the daily vitamin d intake . For some of the food groups (eggs, potatoes / fruits / vegetables, nutriments, meat / meat products, and others), median values for their contribution to vitamin d intake amounted to zero this is due to the very low amount of vitamin d in these food groups and/or the fact that within the 3 days of dietary record, more than half of the subjects consumed no such foods (as in the case of eggs, for example). Median and mean percentage contributions of food groups to the daily vitamin d intake differed considerably, indicating non - normally distributed data . When expressed as mean values, contributions to the daily vitamin d intake amounted to approximately 40% for fish / fish products followed by eggs (15%), fats / oils (13%), bread / bakery products (12%), and milk / dairy products (12%). The other food groups altogether added to less than 10% to the mean daily vitamin d intake . Stratifying our analysis by age (<76 vs. 76 years), vitamin d status tended to be higher in younger subjects compared to older subjects (median: 65.2 vs. 61.2 nmol / l; p=0.053), whereas dietary vitamin d intake was significantly higher in older participants (median: 2.4 vs. 4.1 g / day; p=0.049). Apart from a tendency towards a higher contribution of fish to the vitamin d intake in older participants (35 vs. 44%; p=0.102), contributing food groups did not differ between age groups . No substantial differences in vitamin d intake levels or in relative contributions of food groups were found after stratifying the cohort according to the median vitamin d status (p>0.05). However, a slightly higher contribution of meat / meat products to the dietary vitamin d intake in subjects with a vitamin d status 63 nmol / l compared to subjects who had a higher vitamin d status was noticed (2.05 vs. 2.02%; p = 0.024). After stratifying the cohort into two groups based on the median bmi, vitamin d status of subjects with a bmi 26.8 kg / m was higher than in subjects with a bmi>26.8 kg / m (median: 67.3 vs. 59.3 no significant differences in vitamin d intake or in contributions of food groups were found (p>0.05). Similar results were observed when the cohort was divided according to the sex - specific median% tbf (p>0.05). Subsequent to the separation of the study population with regard to the monthly household net income (<1,500 vs. 1,500), vitamin d status was higher in the upper income group (median: 59.4 vs. 65.6 nmol / l; p=0.020), while vitamin d intake was significantly lower (median: 5.5 vs. 2.7 g / day; p=0.007). Compared to subjects reporting an income 1,500 /month, subjects with an income <1,500 /month showed higher mean fractional contributions to dietary vitamin d intake by fish (49 vs. 36%; p=0.028) and by potatoes / fruits / vegetables (2.4 vs. 2.3%, p=0.044), whereas the contributions of fats / oils (9 vs. 16%, p=0.010) and, albeit not significant, of milk / dairy products (9 vs. 13%, p=0.066) were lower . Dietary vitamin d intake was associated with age,% tbf, intake of energy, fat and alcohol, frequency of fish intake, and household net income, but did not correlate with sex, bmi, smoking, pal, use of vitamin d supplements, and consumption frequencies of eggs and milk . Except for a positive correlation with alcohol intake (r s=0.152; p=0.020), 25(oh)d3 levels did not correlate with dietary parameters (p>0.05). Spearman correlations between vitamin d intake and relevant parametersa r s = spearman correlation coefficient . Missing data were present on total body fat (n=5); consumption frequencies of fish (n=9), eggs (n=6), and milk (n=23); physical activity level (n=26); smoking behaviour (n=2); and monthly household net income (n=41). Frequencies were dichotomised in never to several times per month (coded as 0) versus several times per week to daily (coded as 1). Multiple regression analysis (n=185) was performed with log vitamin d intake as the dependent variable and age,% tbf, intake of energy, fat and alcohol, frequency of fish intake, and household net income as independent variables . Thereby, fat intake (=0.283; p<0.0001) and frequency of fish consumption (=0.235; p=0.001) were identified as positive determinants of dietary vitamin d intake, whereas alcohol intake (=0.122; p=0.082), net income (=0.174; p=0.013), and% tbf (=0.174; p=0.014) negatively affected vitamin d intake . This regression model explained 19.6% of the variance in dietary vitamin d intake in elderly subjects . The exclusion of two people with very high dietary vitamin d intake levels (26 and 37 g / day) provided approximately equivalent results . This cross - sectional study demonstrates that independently living elderly subjects can generally obtain 25(oh)d3 concentrations 50 the facts that this study was conducted in summer and gisela subjects spent on average 2 h daily outdoors are probably the main reasons for this observation (23). A sun exposure of 515 min / day of hands, face, and arms from 10 am to 3 pm in summer is considered as sufficient to provide 1,000 iu of vitamin d (24). Contrary to the present investigation, several studies found a significant association between vitamin d intake and serum 25(oh)d levels (1, 4, 25, 26), but predominantly these studies did not focus on vitamin d status of elderly subjects during summer . In addition, studies were frequently carried out in countries, such as the united states and canada, where food fortification is more common than in germany . A vitamin d intake of 34 g / day as noticed in the present study is apparently too low to influence 25(oh)d3 levels of the elderly in summer, when uvb exposure is the main contributor (8, 23). (25) who found a significant association between vitamin d intake and vitamin d status in 507 free - living subjects aged 6584 years in all seasons except for summer . (27) with 3,113 postmenopausal women, the association between dietary vitamin d and 25(oh)d was significant only in winter and spring . Obviously, vitamin d intake becomes negligible in case of sufficient sun exposure, but it may gain in importance in wintertime, particularly for elderly obese subjects as a higher% tbf has been linked to lower 25(oh)d3 levels (23). Our results indicate that subjects with higher% tbf consume less vitamin d than lean subjects even after taking energy intake and frequency of fish consumption into account . This may further enhance the impairment of the vitamin d status in case of an insufficient sun exposure . In our study, fish and fish products were by far the major sources of dietary vitamin d independent of sex, age, vitamin d status, bmi, and net income . In addition, fats / oils, eggs, bread / bakery products, and milk / dairy products contributed to vitamin d intake . Although innards contain high amounts of vitamin d (6), such foods are not commonly consumed by the general population . In the present study, a representative study of german subjects aged 1480 years provided comparable results by also stressing the importance of regular fish intake as a main source of vitamin d followed by fat spreads, eggs, and dairy products (13), but 25(oh)d concentrations were not investigated . Although regular intake of fish was associated with a higher vitamin d intake in our study, fish intake did not affect vitamin d status . It should be stressed that fish intake of the gisela participants was only slightly below the current german food - based dietary guideline which recommends a weekly intake of at least 150 g of fish (28), but still vitamin d intake was low . This may be due to a higher consumption of lean fish, which contains lower quantities of vitamin d than fatty fish (29). Compared to other european countries, fish intake in germany is rather low (30). However, even in populations with higher fish consumption, frequency of fish intake is not associated with 25(oh)d levels in the elderly (31). In general, the amount of vitamin d in fish varies widely, and often differs from the vitamin d content that is listed in current food composition tables (32). For example, it has been shown that wild - caught salmon contains nearly five times more vitamin d than farmed salmon (33). Regular consumption of fatty fish such as herring and salmon is required to get high amounts of dietary vitamin d. as pointed out by sirot et al . (29), recommendations on fish consumption should refer to species and not only differentiate between lean and fatty fish to take into account differences in contents of nutrients and contaminants . Current german food - based dietary guidelines recommend a higher consumption of lean rather than fatty fish and give no explicit statement on fish species (28). Whether a re - evaluation of the current guidelines could result in an increased vitamin d intake has to be evaluated . Contaminants in seafood and aspects of sustainability of fisheries may argue against an increase in fish consumption . Main vitamin d providing food groups differ among countries . In the united states and canada, milk, meat, and fish are the leading food sources of vitamin d (7, 15), while in the united kingdom, these are fish, meat, cereals, and fat spreads (34). In japan, fish intake is by far the main contributor followed by eggs and mushrooms (35). Differences in assessment methods, dietary habits, and food fortification policies may account for these divergent results (7, 36). In germany, only a few foods are allowed to be fortified with vitamin d including margarine, edible oil, and some diet and dairy products (37). Enhanced food fortification with vitamin d and supplement use whether these recommendations are appropriate for all people independent of their individual risk for vitamin d deficiency is up for discussion . Vitamin d supplements are considered as a simple, effective, and low - cost intervention (38). However, elderly subjects often take many different drugs, so that an additional supplement may not be an appropriate public health strategy regarding compliance and potential interactions . Likewise, an expansion of food fortification polices should undergo a well - considered risk benefit assessment . There are indications that food enrichment cannot ensure adequate vitamin d intake levels for the elderly without putting children at risk (3). Furthermore, lactose malabsorption / intolerance is frequently observed in the elderly (39) and thus milk as a food providing vitamin d seems unsuitable for the elderly population . Our results indicate that a high vitamin d intake level is apparently not required in free - living elderly subjects in summer when sun exposure is sufficient . Hence, a food fortification which is limited to winter months might be appropriate, but may be difficult to implement . A special feature of our approach is that we investigated in a subgroup analysis whether vitamin d intake and vitamin d food sources differ by sex, age, vitamin d status, bmi, or income . Overall, considerable differences regarding the contribution of food groups to the dietary vitamin d intake were not found except for household income . In this context, fish / fish products contributed less to the vitamin d intake of subjects with a monthly household net income of 1,500 compared to subjects who reported a lower income, whereas in the upper income group, a higher percentage of dietary vitamin d was provided by the food category fats / oils. In contrast to some previous studies (15, 16), in which income was a positive predictor of vitamin d intake, household income was inversely associated with vitamin d intake in our subjects . However, despite a lower intake level, serum 25(oh)d3 concentrations were higher in the upper income class, although the self - reported time spent outdoors and the proportion of supplement users did not differ between these two income groups (p>0.05). These observations may be due to the fact that the overall vitamin d intake levels were too low to influence the vitamin d status significantly during the summer . In addition, low income subjects had a significantly higher% tbf than subjects with higher income (42 vs. 38%, p=0.001), which may have masked any potential effect of a somewhat higher vitamin d intake on the vitamin d status of the subjects with lower income, as it was shown that the% tbf is a negative predictor of 25(oh)d3 concentrations (23). Limitations of the present study are the cross - sectional design and the sample size . Although the gisela study is not based on a nationally representative sample, independently living elderly people represent the majority of older individuals in industrialised countries (4). In general, dietary assessment tools have several limitations including the presence of under-/over - reporting and intra-/inter - subject variability of food intake (40). Nutrient databases are frequently not up - to - date, not standardised, and fortified and convenience foods are under - represented (41). In addition, vitamin d intake levels by supplements were not assessed; however, the number of supplement users was small . Concerning the association between net income and vitamin d intake, some subjects gave no statement on their net income, which may have biased the association . Elderly people living in private households do not reach the current recommended vitamin d intake level . Frequency of fish consumption and intake of fat are positive determinants of vitamin d intake, while% tbf and household net income are inversely associated with vitamin d intake . There are no substantial differences in vitamin d food sources depending on sex, age, vitamin d status, and bmi . Income - dependent differences in habitual dietary vitamin d intake are not reflected by serum 25(oh)d3 concentrations in the independently living elderly during summertime . Dietary advice and if sun exposure and dietary vitamin d intake cannot provide sufficient vitamin d concentrations, supplements may be advisable . This investigation received no specific grant from any funding agency in the public, commercial, or not - for - profit sectors.
The problem of finger in the patient with diabetes mellitus is important consideration in diabetology . Generally, peripheral neuropathy that manifests with finger paresthesia, numbness, and blanching is common . Nerve conduction studies, vibration and temperature threshold measurements, and neurovascular function tests are useful for assessment of these cases . However, some recent reports mention the concern on trigger digits in diabetic patients . In this brief article, the authors focus review and discussion on this specific topic . Indeed, trigger digits can be seen in any population . However, some recent publications report on the importance of this disease in diabetes mellitus . The incidence of trigger digits was about four times higher than in the general population . The trigger digits can be seen in 1:20 (cases with trigger digits: all diabetic patients). However, some other reports such as that by aydeniz et al . Show no significant increase in the incidence of this condition in diabetic patients . It is concluded that the trigger digits are an important problem for the diabetic patients . Screening for diabetes may be warranted in patients with involvement of more than three digits . The summary of the important publications reporting on the prevalence of the problem can be seen in table 1. [711] although there is a difference in the rate of reported prevalence and whether the prevalence among diabetic patients is higher than that of normal population or not is still questionable, it cannot be refused for the important of the problem on trigger digits among diabetic patients . Summary on some important publications reporting the prevalence of trigger digits among diabetic patients however, there is no clear evidence that diabetes mellitus increases the risk for development of trigger digits . Although it is not questionable that overall musculoskeletal problems increases in diabetic patients, there is no conclusion on the specific trigger digits problem . Focusing on the existed evidences, trigger digits are common in old diabetic patients but not relating to sex, age, and type of diabetes. [71113] it is of interest that there is an observation that limited joint mobility is related to multiple digits involvement in diabetic patients with trigger digits, but there is no relationship with age, sex, type of diabetes . Although the trigger digits can be seen and similarly diagnosed in both normal and diabetic patients, natural history of the condition in diabetic patients and the outcome of treatment may not be the same . It is reported that the insulin - dependant cases usually have more sever symptoms and multiple digits involvement and require surgical release for relief of symptoms . The steroid injection might be used although it does not provide a good success rate (about 30%). [1719] in addition to use of steroid injection, the use of nonsteroidal anti - inflammatory drugs can provide a little relief from the symptoms. [1719] the use of steroid injection (either methylprednisolone acetate or triamcinolone acetonide) is proved to be safe; however, there is also a report showing that the use of steroid injection can result in hyperglycemia . In addition, the recurrent rate is very high in the diabetes type 1 cases . Focusing in detail, for the surgical treatment, it is used in the severe cases. [172426] the recommended surgical technique is surgical release of the first annular (a1) pulley . The surgery might be a definitive treatment (success rate up to 99%) but the complications can be seen and the postsurgical physiotherapy is still required for a long time . Of interest, a recent report indicated that diabetes was not a risk factor for trigger digits and postoperative complications of trigger digits surgery . However, closed observation and special care are still recommended for the cases with diabetes mellitus due to the risk for existence of microangiopathy . Trigger digits is an important problem in diabetic patients, especially for the old ones . However, the cause result relationship between diabetes mellitus and trigger digits is still the topic for further study . In management, glucose control is important and the standard managements for the general population can be effectively used for the diabetic cases.
The host's immune response is one of the main pathogenic determinants of lung injury during pcp [17] and involves the interactions between immune cells and soluble mediators such as cytokines and chemokines . Animal models of scid, rag1, rag2 or, cd4-t - cell - depleted mice directly demonstrate the role of cd4 t cells in resistance to pneumocystis infection [8, 9]. In humans, the importance of cd4 t cells is demonstrated by the clinical observation that pcp occurs in patients, of 5 years of age and older, when cd4 t cell counts fall below 200 cells / mm . Although cd4 t cells are required for effective host defense against pneumocystis infection, these cells also contribute to immune - mediated lung injury during pcp . For example, pneumocystis - infected scid mice have very little lung damage until the late stages of disease . However, if these animals are immune reconstituted with congenic splenocytes, an intense immune - mediated inflammatory response consisting of cd4 and cd8 t cells is initiated, causing substantially impaired lung function . A similar phenomenon occurs when aids patients receive antiretroviral therapy, or when steroids are tapered in cancer patients receiving chemotherapy [1113]. In patients, this clinical manifestation of pcp is termed immune reconstitution inflammatory syndrome (iris). In murine models of iris, cd4 t cells are most abundant during the acute stage of disease that coincides with the development of impaired pulmonary physiology and organism clearance . The severity of iris is closely related to the extent of cd4 t cell recovery, and these cells therefore, anti - cd3 has been explored as a possible adjunctive therapy for the treatment of pcp . The pan - t - cell antibody anti - cd3 was used in a murine model of pcp - related iris to assess whether it was effective for reducing the severity of pcp even if administered after the onset of disease . Mice that received anti - cd3 antibody exhibited a rapid and dramatic reduction in pcp - associated inflammatory lung injury within 1 week after beginning treatment, and a significant enhancement of survival rate compared with mice receiving control antibody (figure 1). The physiologic improvement in anti - cd3-treated mice was associated with a significantly reduced cd4 and cd8 t cell influx into the lungs . Combining anti - cd3 with trimethoprim - sulfamethoxazole treatment allowed for eradication of the organism as well as control of the associated inflammatory injury . These data suggest that monoclonal antibody - mediated disruption of t cell function may represent a specific and effective adjunctive therapy to rapidly reverse the ongoing pathologic immune response occurring during active pcp . . This experience could provide the starting point for developing a clinical trial of adjunctive therapy for pcp . In contrast to cd4 t cells, cd8 t cells are more abundant during the resolution phase of iris [7, 15], and separate reports by bhagwat et al . And swain et al . Reported that cd8 t cells can exert anti - inflammatory function during pcp by controlling the intensity of cd4 t - cell - mediated immune response [16, 17]. If immune - reconstituted mice are depleted of cd8 t cells, they are able to clear the pneumocystis infection but develop severe inflammatory disease, with increased ifn- production and a prolonged cd4 t cell response compared to fully reconstituted mice . Most often, however, pcp occurs in the setting of persistently low or poorly functional cd4 t cells . In this setting, cd8 t cells contribute to the inflammatory lung injury [7, 18]. In the absence of cd4 t cells, cd8 t cells do not clear the organism but do produce an ineffective immune response that results in significant lung damage and respiratory impairment [16, 18]. Cd8-t - cells mediated inflammation and pulmonary dysfunction are characterized by increased lung production of tnf- and chemokines and altered surfactant homeostasis . While our studies have documented the pathologic role of cd8 t cells during pcp, these cells may also have benefit under certain conditions . Kolls et al . Demonstrated that adenoviral - mediated delivery of ifn- to the lungs of cd4-t - cell - depleted mice resulted in enhanced host defense against pneumocystis infection . The ifn--mediated effects were dependent upon the generation of t cytotoxic-1 cd8 t cells and gm - csf [1921]. Together, these studies suggest that the development of therapies to modulate cd8 t cell phenotype and function could have beneficial effects of pneumocystis clearance and immunopathogenesis . Regulatory t cells (treg) are a subset of t cells expressing cd4, cd25, and foxp3 . These cells maintain immunological tolerance to self and regulate the immune response to infectious organisms . Treg cells represent an important mechanism for the maintenance of immune homeostasis in the lungs, and recent studies have explored the role of tregs during pcp . Showed that adoptive transfer of cd4cd25 t cells attenuated lung inflammation in pneumocystis - infected scid mice . In a later study, mckinley et al . Found that adoptive transfer of cd4cd25 t cells to mice with pcp - related iris reduced pulmonary inflammation and lung injury . These data suggest that cd4cd25 t cells control the immune response during pneumocystis, and modulation of the number and function of this cell population could be a potential therapeutic choice for pcp . Clinical studies in aids patients and other immunosuppressed patients with pcp suggest that disease severity is directly proportional to both neutrophil and interleukin 8 levels in the lung [13, 5]. The association between lung injury and neutrophil influx has also been noted in mouse models of pcp . In the studies by swain, the role of neutrophils in lung damage was studied in several different mouse models with a variety of neutrophil defects . These included: gp91 knockout mice in which production of reactive oxygen species (ros) by neutrophils is greatly reduced gp91- and inducible - nitric - oxide - synthase- (inos-) double - knockout mice in which both ros and no production is greatly reduced; chemokine cxcr2 receptor knockout mice in which neutrophil recruitment to sites of infection is greatly reduced; finally wild - type mice depleted of neutrophils by systemic administration of antineutrophil monoclonal antibody . In each case, despite impaired neutrophil function or reduced numbers in the lung, the physiological parameters of lung injury or the pneumocystis burden were the same as measured in mice with fully functional neutrophils . This study suggested that neutrophil influx into the lung is an indicator of disease severity rather than the direct cause of the lung injury . Therefore, modulation of neutrophil recruitment or function seems unlikely to be effective for the treatment of the immune consequences of pcp . Alveolar macrophages (ams) are a critical component of innate and adaptive immunity in the lungs and are important for host defense against pneumocystis infection . Lebron et al . Reported that pneumocystis -glucan stimulates tlr4-independent nf-b activation in a murine macrophage cell line by using a receptor other than tlr4 which is required for the macrophage tnf- response to lps . Furthermore, steele et al . Reported that murine alveolar macrophages recognize pneumocystis-glucan through dectin-1 pattern recognition molecule, and that, this interaction is required for secretion of mip-2 and nonopsonic phagocytosis . A more recent study by zhang et al . Demonstrated that nf-b is involved in il-8 production by ams in the response to rat pneumocystis . In addition, the mannose receptor is required for the activation of nf-b and il-8 secretion . Studies of tlr2-deficient mice revealed that tlr2 is critical for am inflammatory responses to pneumocystis . These data suggest that ams are important inflammatory mediators during pneumocystis infection and that modulation of am function is a potential strategy to regulate the lung inflammatory response during pcp . Reported that am apoptosis during pcp contributes to the weakened immune status in the lung and promotes the progression of disease . Blockade of am apoptosis with caspase inhibitors enhanced the clearance of pneumocystis organisms in rodent models of pcp, suggesting that modulation of am survival could represent a therapeutic approach to treatment . Much of the recent work related to macrophage biology has focused on the generation and function of distinct polarized, macrophage subsets . During the adaptive immune response macrophages can become activated by antigen - specific th cells, and th - derived signals induce distinct activation programs in macrophages that are directly related to polarity of the th response [30, 31]. Classically activated macrophages (cams or m1) are induced by the th1 cytokines ifn- and tnf-, and they express high levels of proinflammatory and host defense molecules, including inos, ros, tnf-, il-1, il-12, mhc ii, and cox-2 . Cams are critical for host defense against intracellular pathogens, but also contribute to the immunopathology associated with chronic inflammatory diseases . In contrast, alternatively activated macrophages (aams or m2) are induced by the th2 cytokines il-4 and il-13, and counterbalance the proinflammatory cam response . Aams express arginase-1, cd163, mr, tgf-, il-10, and il-1 receptor antagonist . Aams are important for host defense against parasitic infections; they have high phagocytic activity, produce anti - inflammatory cytokines, and are important for tissue repair . We have recently found a striking relationship between host defense, immunopathogenesis, and am phenotype in a mouse model of pcp - related iris . The immunomodulatory drug sulfasalazine (ssz) dramatically reduced pcp - related immunopathogenesis while also enhancing macrophage - mediated phagocytic clearance of pneumocystis organisms from the lung (figure 2). Importantly, the beneficial effects of ssz on immunopathogenesis and phagocytosis were associated with the alternative activation of lung macrophages (m2). This effect may either be the result of ssz promoting a th2 response environment in the lung, or the result of ssz exerting direct effects on ams . This study suggests that alternatively activated macrophages are better suited to deal with pneumocystis infection in a less inflammatory manner than classically activated macrophages . A recent report by nelson et al . Corroborated our finding by demonstrating that m2-polarized macrophages efficiently kill pneumocystis organisms . These studies suggest that ams not only are important for host defense against pneumocystis infection, but also contribute to the lung inflammatory response during pcp . Thus, control of am phenotype with pharmacologic agents, such as ssz, might be beneficial in the treatment of pcp . Lung epithelial cells are the predominant cell types for pneumocystis to interface with in the lung . Importantly, these cells are uniquely positioned in close proximity to blood vessels which carry inflammatory and immune cells that mediate the host response to infection . Studies with both aec cell lines and primary cultures of aecs found that they produce inflammatory cytokines such as il-6 and il-8 and chemokines such as mcp-1 and mip-2 in the response to pneumocystis surface -glucan or live organisms [3436]. Mcp-1, a chemokine involved in lung inflammatory responses and lung epithelial cell repair, is produced by pneumocystis - stimulated aecs through a mapk- and nf-b - dependent mechanism . Furthermore, in vivo studies using tnf receptor deficient bone marrow chimera mice determined that tnf signaling in lung parenchymal cells is important for the inflammatory lung injury during pcp . These studies suggest that aecs are important inflammatory mediators during pcp, and that modulation of aec - specific responses could help reduce pcp - associated immunopathogenesis . Currently, high - dose corticosteroid treatment is utilized as adjunctive therapy for pcp with the intent of reducing the pathological immune response [39, 40]. However, a study by walzer et al . Found that survival rates of patients with pcp in the period before steroids were routinely used as adjunctive therapy was not dramatically different from survival rates of patients in the poststeroids era . Although steroids are effective at controlling the proinflammatory responses of immune cells, they also have proapoptotic consequences for structural cells of the lung, including epithelial cells . It has even been suggested that the proapoptotic effect of steroids on lung epithelial cells contributes to the remodeling associated with asthma and may also exacerbate hyperoxia - induced alveolar injury . This effect may explain in part the failure of pcp treatment in many patients . As discussed above, nf-b activation is involved in both epithelial and macrophage response to pneumocystis infection . Nf-b might facilitate the generation of a successful immune response and prevent the onset of pcp in immunocompetent hosts, while serving to promote and/or amplify immune - mediated mechanisms of lung injury during pcp [35, 36]. Nf-b is an interesting target for therapeutic modulation of immune - mediated lung injury during pcp, as it is involved in the generation of both innate and adaptive immune responses . Specific blockade of nf-b, or certain related signaling kinases, could reduce the pathological immune response associated with pcp and could improve outcome for patients . Ssz is a potent anti - inflammatory drug commonly used to treat the inflammatory consequences of inflammatory bowel disease and rheumatoid arthritis [4345]. Many effects of ssz are related to its function as a potent inhibitor of nf-b, a signaling pathway that is important for initiating inflammatory responses to pneumocystis [49, 50]. Ssz - treated mice had reduced immune cell recruitment, reduced cytokine and chemokine production in the lung, less histological evidence of lung disease, better pulmonary function, and greater survival rates than untreated mice with pcp . As noted above, these data suggest that blockade of the nf-b signaling pathway with agents like ssz can attenuate the inflammatory aspects of pcp, and may warrant further exploration as a viable therapeutic target for the treatment of patients . Other signaling pathways such as mitogen - activated protein kinase (mapk) we found that the jnk pathway mediates mcp-1 production by pneumocystis - stimulated primary murine aec cultures . In addition, carmona et al . Detected erk and p38 activation in human aecs following exposure to pneumocystis -glucan . Blockade of jnk can inhibit proinflammatory gene expression on cd8 t cells and induce apoptosis of cd4 t cells from multiple sclerosis reveals disease patients . Therefore, targeting specific mapk signaling pathways could also effectively reduce the inflammatory consequences of pcp . Granulocyte - macrophage colony stimulating factor (gm - csf) is a growth factor important for monocyte and macrophage proliferation, differentiation, and activation . Gm - csf plays an important role in host defense [52, 53], and mandujano et al . Showed that gm - csf significantly decreased the intensity of pcp infection in a cd4-t - cell - depleted mouse model . This effect is associated with an enhanced tnf- production in ams . In a later study by paine et al ., cd4-depleted gm - csf - deficient mice developed more intense infection and inflammation compared with wild - type mice . Ams from gm - csf - deficient mice demonstrated impaired phagocytic function and reduced tnf- production in vitro . Gm - csf has also been shown to be required for pneumocystis clearance in neonatal mice and for in vitro killing of organisms by tc1 cd8 t cells [21, 56]. Therefore, gm - csf enhancement could be a potential strategy to enhance am function and host defense against pneumocystis . It is critical for the host defense against pneumocystis, but it also plays an important role in pcp - related lung injury through the recruitment and activation of inflammatory cells that amplify pulmonary inflammation and lung damage [7, 15, 57]. Wright et al . Found an inverse correlation exists between lung tnf- levels and pulmonary function in mice with pcp . Furthermore, the severity of pcp is also dramatically attenuated in tnf- receptor knockout mice . However, inhibition of tnf- delayed pneumocystis clearance in normal mice, and neutralization of tnf- in immune - reconstituted pneumocystis - infected scid mice prevented organism clearance . Therefore, any therapeutic regiments that block tnf- signaling to reduce inflammation and lung injury would need to be combined with anti - pneumocystis antibiotics to counteract the negative effects on host defense . Research to date on host immune responses to pneumocystis infection has revealed some aspects of the mechanisms utilized by immune cells that contribute to the pathogenesis of pcp - associated lung injury . The extensive interaction between the immune response and pneumocystis organisms suggests that specific immune modulation could be an effective therapeutic strategy to treat pcp patients . A better understanding of the pathogenesis of this disease may lead to improved outcomes in patients with pcp identifying more efficient and more specific immunomodulation therapies.
Most osteoporotic compression fractures heal with mild residual deformities, and clinical complications are usually rare5). When conservative treatment offers no symptomatic improvement, percutaneous vertebroplasty or balloon kyphoplasty have been safely used and have provided rapid pain relief with mechanical stabilization10). Sometimes, however, patients complain about both back pain and newly developed leg pain following vertebral compression fractures . The main symptom can be radicular leg pain, rather than axial pain, which mimics foraminal stenosis or disc herniation . This radiculopathy is thought to be caused by the encroachment of the intervertebral foramen2). In those cases, it is difficult to perform bone cement augmentation because it is known that percutaneous vertebroplasty is a relative contraindication to the conditions causing radicular symptoms in patients who present with canal encroachment1). This study aimed to investigate the risk for newly developed radiculopathic leg pain following lumbar osteoporotic compression fracture by analyzing the fracture pattern . We also studied whether bone cement augmentation would lead to a good or poor clinical outcome . Between may 2008 and april 2010, 238 patients underwent single - level vertebroplasty or balloon kyphoplasty for osteoporotic compression fractures in our institute . The inclusion criteria were as follows: 1) fracture location under l2 level that can lead to radiculopathic leg pain following vertebral compression fracture; 2) osteoporotic spine (t - score by bone mineral densitometry lower than -3.0); 3) follow - up period longer than 6 months; and 4) no radiculopathic leg pain before the osteoporotic compression fracture . Patients with obvious burst fractures at the time of injury, with previous history of spine surgery or radiculopathic symptom before compression fracture, were excluded in this study . Of these 238 patients, 59 who had osteoporotic compression fractures and underwent percutaneous vertebroplasty or balloon kyphoplasty under the l2 level were enrolled in this study . To study whether the fracture pattern influences the newly developed radiculopathy and back pain, the 59 patients were divided into two groups, namely group a (n=44), back pain - only, and group b (n=15), back pain with newly developed unilateral leg pain . Fractures were evaluated using early magnetic resonance imaging (mri) based on kanchiku's method and we classified into three types according to the main position of the fracture line on the basis of t1, t2-weighted fat and suppression images (superior, middle, and inferior) (fig . The preoperative group differences data were evaluated by the unpaired student t - test using the spss program (spss inc ., chicago, il, usa), and intragroup changes associated with the procedure were evaluated using the student t - test for paired data with a p<0.01 considered the significance level . The anatomical distribution of the 59 patients was as follows: l2 (n=19), l3 (n=18), l4 (n=14), and l5 (n=8). Among them there were no significant differences between group a and group b with respect to age and gender . The overall incidence of radiculopathic leg pain under the l2 level was 25% (n=15). There was no newly developed radiculopathy at the l2 level, and the frequency of radiculopathic leg pain increased with descending levels (table 1). Although vertebral collapse was not severe in lumbar radiographs and mri, 10 of 15 patients in the group b showed compression fracture and suspicious impingement of the corresponding nerve root at the intervertebral foramen . In the analysis of fracture pattern under the l2 level, which showed radiculopathy, only the back pain group (group a) demonstrated symptoms relating to the superior fracture type . However, the fracture pattern in the group with back and radiculopathic pains (group b) was mostly related to the inferior fracture type (table 2). Such a difference was statistically significant (p<0.01) and can be attributed to the different pathophysiological mechanism in the two groups (fig . The mean back pain score of both groups showed significant improvement at the last follow - up . Interestingly, the mean pain score of leg pain in group b also showed significant improvement after bone cement augmentation without decompressive open surgery . The visual analogue scale decreased from a preoperative score of 6.5 to 3.0 at 7 postoperative days and it was maintained at final follow - up with respect to radiculopathy (fig . 3). Three patients (2 in l4 and 1 in l5) in group b had recurrent radicular pain during the follow - up period, but their pain was tolerable, as controlled by analgesics (fig . 4, 5). In the analysis of fracture pattern under the l2 level, which showed radiculopathy, only the back pain group (group a) demonstrated symptoms relating to the superior fracture type . However, the fracture pattern in the group with back and radiculopathic pains (group b) was mostly related to the inferior fracture type (table 2). Such a difference was statistically significant (p<0.01) and can be attributed to the different pathophysiological mechanism in the two groups (fig . The mean back pain score of both groups showed significant improvement at the last follow - up . Interestingly, the mean pain score of leg pain in group b also showed significant improvement after bone cement augmentation without decompressive open surgery . The visual analogue scale decreased from a preoperative score of 6.5 to 3.0 at 7 postoperative days and it was maintained at final follow - up with respect to radiculopathy (fig . 3). Three patients (2 in l4 and 1 in l5) in group b had recurrent radicular pain during the follow - up period, but their pain was tolerable, as controlled by analgesics (fig . 4, 5). Compression fractures caused by osteoporosis are common in the elderly and are often encountered by spine surgeons . It is well known that bone cement augmentation procedures such as percutaneous vertebroplasty or balloon kyphoplasty improve back pain from osteoporotic compression fractures4,8). However, in some instances, lumbar osteoporotic compression fractures without evident canal compromise cause radiculopathic radiating pain and spinal canal stenosis leading to claudication7). Conservative treatment using a lumbar brace for compression fracture forces the posture in lumbar extension, worsening central or foraminal stenosis2). Surgical treatment of lumbar stenosis with open decompression with instrumented stabilization has the potential for significant complications because of comorbidities related to advancing age, especially severe osteoporosis and high risk for instrumentation failure9). This study was performed to clarify and analyze whether an early fracture pattern based on mri classification influences the newly developed radiculopathy in osteoporotic compression fractures and clinical outcome after bone cement augmentation procedure . In this study, the incidences of acute and newly developed leg pain after the possible cause of radiculopathic leg pain after osteoporotic compression fracture may be considered to be the following mechanism: when the osteoporotic vertebral fracture occurs, if the bony fragments from the collapsed body invade the foraminal space, the root can be compressed directly, leading to radiculopathy . The decreased foraminal height by vertebral body collapse will be worse . By categorizing fractures on the basis of fracture line the second plausible mechanism may be considered to be a referred pain, which is distributed far from the site of injury . Doo et al.3) reported that the posterior branches can be irritated by the narrowing of the intervertebral foramen resulting from the decreased height of the vertebral bodies, their pain may have been distributed in these areas as a radicular or somatic referred pain . We performed bone cement augmentation alone because it is a simple and less invasive procedure, and it was efficacious in providing satisfactory pain relief, not requiring open decompressive surgery even in radiculopathy . The pain relief mechanisms by vertebral augmentation procedures may be deduced as the following mechanism . First, segmental stability provided by the cement augmentation is increased, and second, retropulsion and foraminal narrowing are decreased . As spondylotic changes progress in the lumbar spine during aging, the foraminal space decreases with facet hypertrophy, reduced disc height, and foraminal or extraforaminal disc herniations . Actually, the radicular pain in most patients in group b was aggravated in the erect position . Because the posterior wall of the vertebral body inferior to the pedicle constitutes a large portion of intervertebral foramen, the size of the intervertebral foramen will diminish when the compression fracture involves that part of the vertebra . If the root impingement occurs by a combination of spondylotic foraminal stenosis and vertebral fracture, the degree of root compression will increase by weight loading because of the decreased stiffness of the vertebral body . If this is the main mechanism of the patients' symptoms, bone cement augmentation may play a major role in relieving the radicular pain secondary to some degree of fracture reduction and mechanical stabilization . We could achieve favorable clinical outcome by bone cement augmentation procedure alone . Despite our successful results, the authors' proposed pathomechanism is presumptive and is supported mainly by the patient's immediate and symptomatic improvement during follow - up . For a more scientific approach to this subject, experimental data include accurate measurement of the size of the intervertebral foramen according to the weight loading in a fractured vertebra . Experimental study with the finite element model can be a good method that can confirm our results . In the near future, randomized clinical trials for comparative studies in a larger population for a longer period are necessary . By categorizing fractures on the basis of fracture line, we found that inferior - type fractures were more related to the development of radiculopathic leg pain . Bone cement augmentation alone can be a safe and efficient option in patients with painful osteoporotic compression fracture and radiculopathy.
The presence of concomitant cranial nerve signs or facial weakness generally prompts a search for cerebral etiologies such as stroke, neoplasms or inflammatory processes, while it may occasionally be due to a lesion located in the high cervical spinal cord.1 in this concise review, we describe the features of spontaneous spinal epidural hematoma (sseh), which is a rare cause of spinal cord compression and a neurological emergency requiring prompt diagnosis and management to prevent morbidity and mortality.2,3 most sseh patients present with either paraplegia or tetraplegia; however, there are numerous descriptions about acute hemiparesis as an initial manifestation of sseh, which may lead physicians to include an acute ischemic cerebrovascular event as a diagnostic consideration,4 thus leading to the concept that sseh should be included in the large list of stroke mimics . In the following sections, we also discuss various concerns regarding the diagnostic and therapeutic conundrums associated with this disease . Sseh represents a rare spinal emergency, with a frequency accounting for less than 1% of spinal epidural space- occupying lesions.2,3 jackson first described sseh in a 14-year - old female in 1869,5 and the first surgically - treated case was reported by bain in 1897.6 although the introduction of neuroradiological investigations and progress in neurosurgery may lead to a sharp increase in the number of diagnoses of spinal bleeding,7 only approximately 530 cases were included in the largest recent literature search for sseh.8 holtas et al have reported their experience with 13 patients with sseh during a nine - year period in a population of 1.49 million total patients, giving an incidence, on the basis of their population, of approximately 0.1 patient per 100,000 patients per year.9 most of the patients present in their 60s or 70s, but all age groups from six months10 to late 80s have been affected, with a slight predominance of the male sex.2,7 any level of the spinal canal may be involved; the location of the hematoma appears to have a bimodal distribution, with peaks at c6 to c7 and t12,2,8 while the level distribution has been shown to depend strongly on age . The localization of sseh at the lower dorsal and lumbosacral segments under the age of 40 appears to be an exception.2 the hematoma remains limited to a small number of segments (three to four).2,11 the disease - related mortality rate ranged from 6 to 8%, and highly correlated with cervical or cervicothoracic hematomas, especially in patients with cardiovascular disease and those undergoing anticoagulant management.2,11,12 a recent study also revealed that a long hematoma may predict a worse outcome.13 although the precise pathogenesis of the disease remains to be delineated, hemorrhagic disorders due to anticoagulants, thrombolytics and anti - platelet agents, platelet dysfunction, pregnancy, vascular malformation, neoplasms and systemic diseases, such as hypertension and rheumatoid arthritis, have been considered as predisposing factors, while many cases without any known underlying cause also have been demonstrated.2,1214 approximately 3% of patients are hypertensive; however, a relationship between hypertension and the development of a hematoma is considered to be marginal.2 nevertheless, the clinical significance of these previous findings should be evaluated carefully in terms of the fact that there is still no established definition of sseh . A spontaneous hematoma is most often defined as a condition occurring in the absence of any traumatic event or iatrogenic procedure,15 and thus, some of the predisposing factors described above cannot be excluded . When it is of idiopathic origin.16 regardless of the etiological background, the actual origin of spinal bleeding has been the subject of some discussion . Despite the presence of literature in support of both venous and arterial origin,17,18 the posterior epidural venous network is believed to be the most likely source of the hematoma, due to the predominance of posterolateral hematomas, the segmental distribution of sseh and the anatomical characteristics of the internal vertebral venous plexus, in addition to the fact that the spinal epidural veins have no valves and are thus prone to damage by changes in abdominal or thoracic pressure.2,12,15 in a recent case series analysis, more than half of the patients with sseh were reported to have experienced a subjective straining - associated event during the initial attack, lending even more credibility to the venous etiology theory.14 on the other hand, beatty and winston made a radical argument for an arterial source of hemorrhage, at least in the cervical region, and they focused on the fact that the intrathecal pressure is higher than the venous pressure at the cervical level, which would preclude bleeding in the intrathecal space from veins.18 the onset of sseh may be associated with neck or back pain radiating to the corresponding dermatome, which may sometimes be vague and ignored until the subsequent cord compression and neurological deficits arise.13 most patients present with paraplegia or tetraplegia,5,11,19 while hemiparesis is considered to be a rare feature of sseh.11,19 due to the rarity of the disease, no prospective series from single departments or study groups are available to date, and the international medical literature, including incidental cases from one s own department, is the only source of information for evaluating this topic.8 more than two decades ago, anderson et al reported that the association with hemiparesis is low, occurring in only 6 of more than 250 reported cases.20 however, modern radiological imaging modalities may overcome the shortfalls of clinical examinations in the management of acute spinal cord injuries, and may lead to new findings . Following the first report published on the use of magnetic resonance imaging (mri) in the diagnosis of acute sseh in 1987,21 the mean incidence of such cases increased from 2.2 to 6.4 new cases per year.22 therefore, the precise incidence of hemiparesis among the overall patients with sseh must be evaluated carefully . Indeed, anecdotal information regarding cases with hemiparesis as an initial presentation is still being accumulated . 4,14,19,2333 the majority of hematomas in such cases were located within the cervical region, while four patients with hematomas that ranged from the cervical to thoracic region were also reported (table 1). The presence of a spinal epidural hematoma was promptly diagnosed in more than half of these cases during the observation period . Persistent neck or back pain and fluctuating neurological symptoms should result in a high index of suspicion of the illness . However, it is noteworthy that there were at least twelve patients who were suspected to have an ischemic stroke during the initial assessment,4,23,25,27,2933 and thus, there were four patients whose neurological manifestations were falsely attributed to ischemic cerebrovascular events who were consequently subjected to anti - coagulation with heparin,25,27 thrombolytic treatment with a recombinant tissue plasminogen activator29 or treatment with an anti - platelet agent as an adjunct to warfarin,23 prior to the final diagnosis that their neurological deficits were etiologically linked to the cervical spinal epidural hematoma . In these cases, the presence of neck pain with or without shoulder pain might have been inadvertently dismissed during the initial physical assessment . Alternatively, or in addition, the occurrence of concurrent dysarthria, 29 which has been demonstrated to be a potential sign for discriminating stroke mimics from actual ischemic stroke in emergency settings,34,35 might also have played a role in the false attribution of the illness to a cerebrovascular event.1 the main cause of dysarthria in the patient with hemipareic sseh described by son et al.29 is unclear . However, they concluded that dysarthria is a rather subjective symptom of patients with stroke, and they noted that removing the patient s dentures during the acute phase of the disease might have resulted in the slurring of speech.29 finally, the hemorrhagic risks associated with anticoagulants may not be equivalent to those of a thrombolytic agent and antiplatelet agent; however, a previous case presentation demonstrating the expansion of an already developing cervical spinal epidural hematoma by the addition of heparin, based on a presumptive diagnosis of a cardiac ischemic event,36 suggests that the clinical picture of patients with sseh mimicking an ischemic stroke might be modulated by the agents described above . The mainstay of treatment for sseh has been surgical evacuation, combined with prompt decompressive laminectomy . There have been several studies that have provided valuable information regarding the outcome following the surgical management of sseh.1113,37,38 based on these findings, the degree of preoperative neurological deficit and the interval between the onset and surgery have been considered to be the critical factors determining the qualitative postoperative recovery.12 incomplete deficits, compared to complete deficits, should be associated with better outcomes . Indeed, the one - year complete recovery rate has been demonstrated to be approximately 89% for patients with incomplete deficits, but only 37.5% for those with complete deficits,11 and similar trends have been demonstrated numerous times in the literature.12,38 in addition, it is necessary to focus on the timing of surgery . In patients with a complete deficit, it has been shown in an analysis of 35 patients with sseh that a good neurological recovery can be achieved after prompt surgery when the time interval from initial ictus to surgery is less than 48 hours, and when the duration of the complete neurological deficits is less than 12 hours.11 another study has demonstrated that a significantly better outcome was obtained if surgical decompression was carried out within 36 hours in patients with complete sensorimotor loss, and within 48 hours in those with incomplete deficits.12 in a series of 14 patients reported by shin et al, the patients who were surgically managed within 12 hours after the onset of symptoms scored significantly higher (84%) on the recovery scale of the japanese orthopedic association39 than those who underwent an operation from 12 to 24 hours after the initial attack (63.6%) and those who underwent surgery more than 24 hours after onset (46.7%).38 similar trends were also noted by lawton et al.37 the postoperative mortality rate of sseh can thus be expected to range from around 3% to 6%.11,37 on the other hand, conservative management may still be an important treatment option in some subsets of sseh patients with mild, rapid and spontaneously recovering neurological deficits.8,40 groen et al recently demonstrated that 84% of patients with sseh treated non - surgically recovered completely.8 however, this finding should be evaluated carefully in terms of the severity of the neurological deficits manifested by the subjects in their study . Indeed, as they stated, the subjects managed conservatively had less severe signs and symptoms and were more likely to be diagnosed based on an imaging modality alone, compared to the surgically - treated cases reported in the literature.8 moreover, a concurrent high surgical risk, such as bleeding diathesis, may also be a determinant in the decision for conservative management.8 it is not surprising that some patients with minimal neurological signs may respond successfully to conservative management; however, the presence of cases in which an initial recovery was followed by deterioration requiring surgery should be kept in mind, and thus, the close observation of such patients should be carried out in a hospital with a neurosurgical specialty team.40 in the ordinary clinical setting, acute stroke patients almost never contact a neurologist initially, since most communities do not have an academic medical center with a systemic neurological assessment program.41 it is known, however, that neurological complications contribute to morbidity and mortality in a wide range of patients,4244 and an accurate recognition of the condition is required to ensure prompt transfer to an appropriate treatment unit . Moreover, there is a limited time window for the administration of a thrombolytic agent, which is the only available medical treatment for acute ischemic stroke that has been proven to be effective.45 in this regard, not only emergency medical services and emergency room departments, but also primary care physicians, should be familiar with the wide range of neurological problems that can develop in order to assure the optimal management of patients . We feel that an early and accurate diagnosis, as well as awareness of sseh, remains a challenge for physicians, despite the accumulation of studies disclosing the nature of the disease . The current concise review emphasizes the pitfalls of evaluating patients with acute hemiparesis due to sseh . The imaging analyses routinely applied for patients with acute cerebral ischemia do not rule out spinal bleeding, while the higher a cervical sseh extends, the more likely it is to be seen on non - contrast computed tomographic scans.4 the sudden onset of neck and/or back pain and progression of hemiparesis to paraplegia or tetraplegia during the observation period are clues leading to a timely diagnosis of cervical spinal lesions.4,19,24,27,3032 although there may be limited access to mri,46 negative brain mri findings, which strongly suggest the absence of ischemic cerebrovascular events,47 may be an alternative trail turning the attention towards other regions, including the cervical spine . In addition, the presence of concurrent broun - squard syndrome, characterized by the ipsilateral loss of proprioceptive sensitivity with the contralateral loss of pain and temperature sensitivity, can help to promptly diagnose the disease,4,4851 although this syndrome is not necessarily associated with hemiparesis as an initial neurological presentation.52 stroke mimics may account for 20 to 25% of suspected stroke presentations,53 and there are various conditions (table 2) that have been demonstrated to act as stroke mimics in previous studies.5457 there are some discrepancies in the distribution of diagnoses, depending on the context, while seizure seems to be one of the major common denominators, being present in approximately 15 to 20% of the stroke mimic cases.5457 some studies included the presence of cervical or spinal lesions in the conditions that mimicked stroke;54,56 however, the lack of information regarding the disease spectrum precludes us from determining the actual frequency of sseh among subjects with a condition mimicking stroke . Nevertheless, we believe that sseh, especially in the cervical region, should also be included as such a clinical entity,4 thereby leading to a high index of suspicion, prompt recognition and immediate intervention, which is essential to reduce or prevent major morbidity of the disease.36 several studies have demonstrated the safety of thrombolytic treatment in some subjects with stroke mimics,58 although there has been only a single report regarding a patient with sseh mimicking a stroke being administered a thrombolytic agent as an initial treatment, and this was followed by urgent laminectomy without any bleeding complications.29 considering the narrow window for thrombolytic treatment in cases of ischemic stroke and the rarity of sseh, one may argue that our proposal is not necessarily justified . On the other hand, the association between the administration of a thrombolytic agent and the secondary development of spinal epidural hematoma has been demonstrated anecdotally.59,60 moreover, the inappropriate administration of agents used for restoring cerebral blood flow may preclude prompt surgery for sseh, due to the patient s modified hemostatic nature, although the information currently available may not necessarily support this concept . 29 thus, we believe that it is necessary to take a proactive approach by adding sseh to the list of differential diagnoses of ischemic stroke before fatal outcomes accumulate.36 finally, it should be kept in mind that we are always facing, as do most physicians at various times, diagnostic and therapeutic dilemmas, and carefully weighing all of the options and potential outcomes on a case - by - case basis is therefore essential.
Because of variations in pathological and clinical features of the disease, npd is classified into those with deficiency of acid sphingomyelinase activity (types a and b), and those with defective intracellular processing and transporting of ldl cholesterol, which is known as type c. however, npd type c (npc) is different at the biochemical and molecular levels with a higher incidence than npa and npb . Both types a and b are characterized by acid sphingomyelinase (asm) deficiency that leads to excessive sphingomyelin accumulation in all phagocytic cells and in neurons . Normally, the enzyme activity of asm leads to the breakdown of sphingomyelin into ceramide and phosphorylcholine in lysosomes . Generally, multiple organs such as spleen, liver, bone marrow, lymph nodes, lung and central nervous system are affected . Clinically, npa is distinguished by failure of thrive, hepatosplenomegaly and progressive neurodegeneration . At molecular genetics level, the smpd1 gene is approximately 5 kb long and the coding sequence is divided among six exons . According to the human gene mutation database (www.hmdb.cf.ac.uk), more than 100 different mutations (including missense, nonsense, deletion, insertion, and splice site mutations) have been reported in the smpd1 gene . To date for instance, motamedi et al reported a case of npb in iran and described the clinical course of his patient . Furthermore, motamed et al reported a ten year study of liver biopsies in children's medical center in tehran . Most identified patients were affected with npd and glycogen storage disease type 1 (gsd - i). In addition, majidzadeh et al reported a case of npd and compared clinical and molecular aspects of the disease . Hoshmand et al presented a novel mutation in the smpd1 gene (data not published). Here, we present the first molecular genetics diagnosis of npa in an individual from southwest iran . A 2.5 year - old male, the first born child from first cousin parents was attending our center for genetic counseling and genetic diagnostics . The patient showed typical signs of the niemann pick disease, such as hepatosplenomegaly, developmental delay, mental retardation, and foam cells in the bone marrow, hypotonia, and cherry red maculae . Unfortunately, measurement of the asm activity in the white blood cells failed and dna test was the only chance to establish a definite diagnosis . After obtaining informed consent, genomic dna was extracted from edta - anticoagulated whole blood by standard salting out procedures . Selective amplification of all 6 exons was performed in a volume of 25 l reaction containing 10 pmol of each primer (tag copenhagen a / s, fruebjergvej3, denmark) and 50ng of genomic dna . Pcr was carried out by a set of designed primers using primer3out software (http://frodo.wi.mit.edu/primer3/). Exon 2 was amplified by two separate primer pairs to assist subsequent sequencing reactions temperature profile of reactions were as follow: initial denaturation at 95c for 3 min, 35 cycles of 95c for 30s, 56 - 60c for 30s and 72c for 45s and a final extension at 72c for 7 min . Direct sequencing of pcr products was carried out using abi automated sequencer 3700 according to the manufacture's instruction (abi 3700, pe applied biosystems, foster city, ca, usa). Sequence analysis was performed with the software bioedit (version 7.0.5.3). According to the human reference sequence nm_000543.4, a homozygous single guanine deletion, c.740delg, was observed in exon 2 of the smpd1 gene, which results in a premature stop codon (ctg> tga) between codons 256 - 257 (p.gly247alafs*9) (fig . They are also obligate carrier . To confirm the pathogenic relevance of the detected deletion, 27 healthy individuals were tested . Chromatogram of sequencing reactions from a) affected individual, b) heterozygote parents, c) healthy person . The location of the novel deletion is indicated with black arrow and the position of the resulting stop codon is shown in black circle on the chromatogram . At bottom (d), partial sequence of the smpd1 gene with corresponding codons is illustrated . To our knowledge by intensive literature searching, the present report demonstrates firstly the molecular genetics diagnosis of the npd in southwest iran . Approximately 1 per 40,000 people of ashkenazi jewish descent has npa . In contrast, there is no information about frequency and distribution of different types of npd in iran, particularly in southwest iran . The disease refers to a group of disorders with deficiency in lipid storage, causing accumulation of fats in brain and liver that lead to serious damage or dysfunction of mentioned tissues . However, liver enlargement, brain damage, difficulty in walking, speaking and learning are characteristic symptoms for npa . Diagnosis of this type of disease is usually made by measuring the asm activity in white blood cells . He was also referred to our molecular diagnostic center with the aim to find genetic cause of the disease . The smpd1 gene is the only gene considered to be associated with npa . Here, we report an unreported deletion of single guanine in exon 2 of the smpd1 gene, causing a truncated gene product . Exon 2 is unusually large, which encodes for 258 amino acids or approximately 44% of the entire asm polypeptide . However, the pathogenic nature of the novel deletion was confirmed by screening of healthy individuals . Three common missense mutations account for more than 90% of the mutant alleles in individuals of ashkenazi jewish ancestry with npa . But in contrast to the ashkenazi jewish population, most affected individuals with npa showed in previous studies unique mutations in the smpd1 gene . Consequently, identification of new npa cases increases the probability to discover new specific mutations for the given population . The present observations extend the mutation spectrum of the smpd1 gene in npa patients worldwide, and can be used for the prenatal or preimplatation genetic diagnosis, at least in southwest iran . Therefore, prevention seems to be more effective strategy in the next pregnancy for this family and other families with positive history.
A 7-year - old spayed domestic longhair cat from perth, western australia, presented with left - sided head tilt, dysphonia, head shaking, inappetence and weight loss . Otitis media with extension into the external ear canal was suspected and investigated using video - otoscopy and computed tomography examination . Invasive disease with extension from the middle ear to the base of the skull, and intracranial extension into the caudal fossa and cranial cervical vertebral canal was detected . Cytology of external ear canal exudate showed capsulated budding yeasts and cryptococcus gattii vgii was cultured . Treatment with amphotericin b infusions and oral fluconazole was prescribed, with nutritional support via oesophagostomy tube . This case report describes the successful medical treatment of otogenic meningoencephalomyelitis due to c gattii (vgii) infection in a cat . Cryptococcosis is a common systemic mycosis affecting cats worldwide and can affect cats of any age and sex . The two most common species causing disease in animals are cryptococcus neoformans and cryptococcus gattii . Historically, cryptococcus was classified into five serotypes (a, b, c, d and ad) on the basis of antigenic differences in capsular polysaccharides . Using a modern molecular typing scheme, cryptococcal strains are now divided into eight multilocus sequence types (vni, vnii, vniii, vniv, vgi, vgii, vgiii, vgiv), which are likely to be cryptic species . C gattii has been associated with tropical and subtropical climates, with an outbreak of disease affecting both humans and animals in the pacific northwest of the usa, emanating from an initial focus of infection on vancouver island, british columbia, canada . C gattii primarily infects immunocompetent hosts and the organism can cause disease in unusual host species (eg, horses, ferrets, goats, dolphins). Vgii has a high environmental presence in perth, western australia, and areas of the northern territory, in contrast to eastern australia where vgi isolates predominate . The genetic diversity of vgii environmental and clinical isolates obtained from the perth environs suggests there is a sexually recombining population structure in this geographical niche . While c gattii vgi is strongly associated with detritus in mature eucalyptus tree hollows, the ecological niche of vgii is largely unknown . In vancouver island, where an outbreak of vgiia was diagnosed in humans and animals, c gattii vgii colonises coniferous, evergreen and deciduous trees in the douglas fir tree bioclimatic zone, with transient isolation from soil, saltwater, freshwater and air . Infection most likely occurs subsequent to inhalation of airborne infectious propagules such as basidiospores or desiccated yeast cells with clinical signs of sneezing, stertor, nasal deformity and discharge reflecting nasal cavity infection . It is common for the infection to spread, both locally to contiguous adjacent structures and haematogenously to other sites . We present an unusual case of otogenic infection with intracranial extension attributable to c gattii vgiib in a cat for which signs resolved with aggressive medical therapy . A 7-year - old spayed domestic longhair cat was presented to murdoch university veterinary hospital for evaluation of left head tilt, dysphonia, head shaking, inappetance and weight loss of 2 weeks duration . Initial investigations by the referring veterinarian identified an aural mass in the left horizontal ear canal, which had been removed . Sabouraud s dextrose agar culture of an ear swab had resulted in heavy growth of c gattii . Serum latex cryptococcal antigen agglutination test (lcat) was positive, with a titre of 1:256 . The cat was treated with enrofloxacin (5 mg / kg po q24h [baytril; bayer animal health]), itraconazole (10 mg / kg po q24h [sporanox; janssen pharmaceutica]), meloxicam (0.05 mg / kg po q24h [meloxicam; troy laboratories australia]) and a topical compounded enrofloxacin / dexamethasone otic preparation . The cat was thin (body condition score [bcs] 3/9; weight 4.9 kg) and had a left - sided head tilt . The remainder of the clinical examination, including full ophthalmic and neurological examinations, was unremarkable . The presence of a head tilt without ipsilateral postural reaction deficits was consistent with left peripheral vestibular disease . Hand - held otoscopic examination demonstrated a mass occluding the lumen of the left horizontal ear canal, and ceruminous discharge . The cat was premedicated with buprenorphine (0.018 mg / kg i m [temgesic; symbion pharmacy services]) and acepromazine (0.04 mg / kg i m [acp; westralian holdings]), anaesthetised with propofol and maintained with isoflurane in 100% oxygen . A ct (siemens emotion duo 2 slice ct scanner; ge healthcare australia) examination of the head was performed using soft tissue and bone spatial reconstruction algorithm and 1 mm slice thickness pre- and postintravenous contrast (iohexol; 10 ml [300 mmol / l] iv) (figure 1), followed by video - otoscopic examination . (a) the medial portion of the left external ear canal is filled with strongly and homogeneously contrast - enhancing soft tissue attenuating material (1). The left tympanic bulla is filled with mildly and homogeneously contrast - enhancing material (3). (b) the material seen ventromedial to the tympanic bulla continues caudally and forms a 1.6 cm (width) 1.3 cm (height) 2.5 cm (length) soft tissue structure, poorly enhancing centrally but strongly enhancing peripherally (1). This mass () distorts local tissue architecture such that the nasopharyngeal lumen is narrowed> 50%, and the hyoid bones are displaced laterally . Additionally, at this level, there is a rim of contrast enhancement seen in the ventral aspect of the left temporal lobe of the cerebrum (2). (c) at the level of the caudal aspect of the left tympanic bulla, the soft tissue lesion can still be seen ventromedial to the bulla (1). Additionally, there is a rim of contrast enhancement outlining a hypoattenuating area in the left lateral cerebellum (2). There is poorly contrast - enhancing soft tissue attenuating material in the caudal fossa that is displacing the brainstem dorsally and to the right (3). Additionally, there is strong meningeal contrast enhancement in this region . (d) the poorly contrast - enhancing material in the cranial cavity can be followed further caudally, to the level of the atlas . This image, at the level of the foramen magnum, demonstrates abnormal tissue (arrow) displacing and compressing the cervical spinal cord () dorsally and to the right . (e) bone window computed tomography image at the most caudal aspect of the bulla . Note the discontinuity of the temporal bone (black arrows), indicative of bony lysis ct showed moderate contrast enhancement of the left ear canal lining . The medial portion of the left external ear canal and left tympanic bulla were filled with homogenous, contrast - enhancing soft tissue attenuating material (figure 1a). Ventromedial and rostral to the tympanic bulla, a region (1.6 cm width 1.3 cm height 2.5 cm length) of poorly contrast - enhancing tissue, outlined by a rim of strong contrast enhancement (figure 1b) causing a mass effect, was seen, which compressed the nasopharyngeal lumen by> 50% and displaced the hyoid bones laterally (figure 1b). Ventromedial and caudal to the bulla, a similarly enhancing region was seen (0.6 cm height 0.3 cm width 0.6 cm length). In the caudal left bulla, there was bony lysis of the temporal bone (figure 1e). Lateral to the oval foramen and dorsal to the larger parapharyngeal lesion, an intracranial rim of contrast enhancement was seen in the ventral aspect of the left temporal lobe . Adjacent to the lytic region of the temporal bone, contrast enhancement outlining a hypoattenuating area with broad dural base was seen (figure 1c). Poorly enhancing material extended caudally along the left ventral portion of the brainstem to mid first cervical vertebra, displacing and compressing the brainstem and cranial spinal cord, accompanied by strong meningeal contrast enhancement (figure 1d). Video otoscopy (medrx video otoscope) identified a large pink fleshy mass obscuring the lumen of the horizontal ear canal . The mass traversed the tympanic membrane, and was contiguous the tympanic bulla (figure 2), in agreement with ct findings . Video - otoscopic photograph of the left tympanic membrane, which appears to have been breached by abnormal inflammatory tissue (arrows), which extends into the tympanic bulla impression smears of the mass revealed large numbers of round yeast, surrounded by a prominent clear halo and narrow - necked budding consistent with cryptococcal infection . A heavy pure growth of a cryptococcus species resulted, which was identified as c gattii by conventional phenotypic mycology testing . Further analysis by sequencing of the ribosomal internal transcribed spacer region identified c gattii vgii by comparing the sequence with published signature sequences; however, the isolate was not available for further molecular typing . The isolate was susceptible to fluconazole, itraconazole, posaconazole, flucytosine and amphotericin b, but resistant to caspofungin . The minimum inhibitory concentration (mic) for fluconazole was 8 mg / l (table 1). Antifungal susceptibility data for the cryptococcus gattii vgii isolate cultured from the cat s = susceptible; r = resistant; mic = minimum inhibitory concentration antifungal therapy was commenced with fluconazole (10 mg / kg po q12h [symbion pharmacy services]) and amphotericin deoxycholate (0.5 mg / kg; subcutaneous infusion in 350 ml 0.45% nacl and 2.5% dextrose three times weekly). Four weeks after treatment commenced, the cat was brighter, with complete resolution of the head tilt, although inappetence persisted ., the cat had received a cumulative amphotericin b dose of 12.5 mg / kg, and was starting to eat and gain weight . Owing to finances, the frequency of amphotericin b infusion was reduced to twice weekly (dose 0.7 mg / kg / infusion). The frequency of amphotericin b infusion was reduced to once weekly (0.7 mg / kg / infusion). Four months after diagnosis, lcat was 1:256 and the plasma concentration of fluconazole was 45 mg / l . Seven months after diagnosis, the lcat was 1:64 and was 1:32 a further 3 months later, with a corresponding plasma concentration of fluconazole of 47 mg / l . After 12 months, the weekly amphotericin b infusions (0.7 mg / kg) were discontinued and the cat continued to be clinically well, receiving only fluconazole (50 mg q12h). At the time of writing, 21 months after starting therapy, the lcat was 1:8 and the cat continued to be clinically well . Otitis media due to c neoformans var grubii infection was managed by a total ear canal ablation and lateral bulla osteotomy, followed by oral itraconazole . In the third cat ventral bulla osteotomy and twice weekly amphotericin b subcutaneous infusions, with oral flucytosine and fluconazole, were used . In these latter two cats, diagnosis was made on cytological and histological evaluation of material removed via bulla osteotomy, culture and serum antigen testing . The cat presented initially with unilateral peripheral vestibular disease . As a mass in the left external ear canal was detected during preliminary examination, an aural polyp arising from the middle ear was initially suspected . Presence of additional signs, including dysphonia, inappetence and weight loss, may have suggested a more sinister underlying aetiology . Cytology from the external ear canal resulted in a prompt diagnosis of cryptococcosis; however, failure to improve clinically to azole monotherapy prompted referral . Video otoscopy and ct of the head demonstrated extensive and invasive disease of the ear, skull, cervical spinal cord and ipsilateral intracranial dural space . Inhalation of air - borne basidiospores or desiccated yeast cells into the nasal cavity is considered the primary route of infection in cats . Colonisation of the caudal nasal cavity, with subsequent epithelial invasion, possibly in the vicinity of the nasopharynx, may have been the primary route of infection, with extension via the auditory tube to the middle ear . Possible pathways from the inner ear into the brainstem include erosion through the medial aspect of the petrous temporal bone (although this was not apparent in the ct scans), along the nerves and vessels of the internal acoustic meatus, or via haematogenous spread . Inoculation of cryptococcal organisms directly into the external ear canal, with subsequent spread to the middle ear by penetration of the tympanic membrane is considered unlikely and usually results in only superficial lesions . A grass seed heavily contaminated with cryptococcal cells may provide an explanation for how this might have occurred, although these are less commonly found in the ear canal of cats than dogs . This case is quite distinct from a recent feline case of bilateral cryptococcal otitis interna, which was thought to have arisen haematogenously as there was no evidence of rhinosinusitis disease or otitis media, and a small cryptococcal lesion was also present in the thalamus; this patient was infected with c neoformans var grubii . The pathogenesis of cryptococcus in this cat is unusual and supports the contention by sykes and malik that vgii and vgiii infections are more invasive than c gattii vgi and c neoformans var grubii infections . All clinical signs resolved after aggressive medical therapy and initial de - bulking within the ear canal . While recent papers concerning c gattii vgi infections of the nasal cavity and contiguous tissues in koalas have emphasised the need for surgical intervention and/or intralesional therapy because antifungal agents may not penetrate well into poorly perfused tissues, the requirement for this probably should be made on a case - by - case basis . C gattii vgii can be divided into vgiia, vgiib and vgiic strains, with vgiia being the major genotype in the vancouver island outbreak . Determining the specific biotype is critical because c gattii vgii and vgiii isolates tend to demonstrate heteroresistance to fluconazole . The epidemiological cut - off values for fluconazole in vgii and vgiii infections are therefore higher than for vgi and c neoformans var grubii isolates . In this instance the causal vgii isolate was susceptible to fluconazole in vitro, with a mic of 8 mg / l . Given the pharmacokinetics of fluconazole in the cat, a dose of 10 mg / kg po q12h should produce peak blood concentrations of fluconazole at steady state in the order of 2080 mg / l . In eastern australia, about 20% of cryptococcosis in cats and dogs is caused by c gattii (predominantly vgi). In western australia, vgiib (mating type) is the predominant species of c gattii, although vgi and vgii (mating type a) also occur . Studies on the c gattii vgiia isolates from the vancouver island outbreak and vgiib isolates from australia have demonstrated that such strains are highly virulent in experimental infections of mice and rats . Although central nervous system (cns) involvement is an important negative prognostic indicator in feline cryptococcosis, this cat improved markedly with combination therapy using amphotericin b and fluconazole . Cross - sectional imaging was not often undertaken when cases of cryptococcosis were first recorded in the veterinary literature . In this case, clinical findings and plain radiographs would not have provided indication of the extensiveness of the disease, and the success of medical therapy in this cat testifies that good outcomes may occur despite extensive invasive disease . Treatment for cns cryptococcosis is prolonged and should be continued until the lcat titre reaches zero, which may take 12 years . In recovered cats, the titre can be monitored every 36 months to allow early detection of recurrences, which is more common than re - infection with a new strain or antifungal resistance . This case report describes the successful medical treatment of otogenic meningoencephalomyelitis due to c gattii (vgii) infection in a cat.
Parkinson disease (pd) is a progressive neurodegenerative disorder with widely heterogeneous manifestations, including motor, nonmotor and neuropsychiatric symptoms . It affect~1% of the people older than 60 years.13 the major pathologic feature of pd is the loss of dopaminergic neurons in substantia nigra pars compacta and, consequently, basal ganglia.4,5 sex differences in prevalence, clinical presentations and severity of pd have been reported in some previous studies.69 although the exact sources of these differences in pd are unknown, sex hormones, genetic factors and variations in dopaminergic pathways have been proposed as possible underlying reasons.1014 identifying sexual variations influences prevention, therapeutic strategies and understanding of sex - related neurobiological differences.15,16 exploration of sexual dimorphism in neurologic diseases such as pd has been recently announced as a research need.3 while studies are unanimous about the higher prevalence of pd among men, there are conflicting data regarding sex differences in clinical manifestations, progression and treatment outcome.1723 motor presentations (ie, tremor and dyskinesia), psychological manifestations (especially depression), sleep behavior disorders and cognitive impairment have been reported as potential aspects of sexual dimorphism in pd.1723 however, data are still controversial, inconclusive and even conflicting in some aspects . As an example, a recent randomized clinical trial did not find any sex differences in daily activities and main motor features.24 in addition to lack of comprehensive data, these controversies can be partly due to interrelationship and confounding effects of clinical and demographic variables.3 some clinical features that were previously reported to be sex related failed to show any difference between males and females with pd when controlled for other demographic and clinical variables . For instance, cognitive impairment was reported to be more common in men;9 however, the difference was much less pronounced when the effect of age, level of education and disease severity were taken into account.23 in another large study, univariate analysis showed that daily activity was more impaired in females, but multivariate analysis revealed no significant sex difference.24 having collected comprehensive data on different pd- related and general features, we aimed to further investigate sexual dimorphism in pd . Our objective was to determine independent differences in clinical manifestations and subtypes, psychosocial functioning, quality of life (qol) and its domains between males and females with idiopathic parkinson s disease (ipd) using a powerful statistical approach . Our a priori research hypothesis was that females with pd experienced more severe nonmotor manifestations, which would also influence their qol and psychosocial functioning more prominently than males . This study was conducted on 157 consecutive patients with ipd from an outpatient referral movement disorders clinic in tehran, iran, between october 2011 and december 2012 . This was a collaborative project between iran university of medical sciences (tehran, iran) and karolinska institute (stockholm, sweden). The study protocol was approved by the ethics committee of the neurology department at firoozgar clinical research development center (fcrdc; affiliated to iran university of medical sciences). Prior to the launch of the study, all patients were informed about the aims and procedures . All participants provided their verbal informed consent to participate in this study . Since the project was designed as an observational research, verbal form of consent was approved by the aforementioned ethics committee . Participation in this study was voluntary, and the patients were free to withdraw from the project whenever they decided . Furthermore, the identity of research participants was protected, since the data files were anonymous and all names were omitted . Recruited patients fulfilled the following inclusion criteria: diagnosis of ipd based on the uk brain bank criteria,25 which was assessed by the same neurologist who was specialized in movement disorders for all participants; current age of 30 years or older and motor disability in the mild - to - severe range but not in the advanced stages that needs wheelchair or hospitalization according to the hoehn and yahr (h&y) criteria (stage <5).26 patients with moderate to severe dementia were excluded from the study, as were those with atypical parkinsonism, including multiple system atrophy (msa), progressive supranuclear palsy (psp) and vascular or drug - induced parkinsonism . Data collection was performed through face - to - face interviews with eligible patients by a trained group of medical interns by means of validated questionnaires and checklists . Patients were also examined for clinical assessments and diagnosis by one neurologist specialized in movement disorders . The demographic checklist consisted of baseline variables (age and sex), educational status, history of smoking, comorbidities, duration of pd (time passed from diagnosis) and history of levodopa administration . Total comorbidity burden was calculated by summing up the number of chronic comorbid conditions in each participant, including depression, hypertension, interstitial heart disease (ihd), diabetes, stroke / transient ischemic attack (tia) and osteoporosis . Data were collected based on participants self - reports and their medical records at the referral center . Clinical characteristics of pd were assessed using the following scales and/or definitions: motor severity: unified parkinson s disease rating scale (updrs) subscales i iv, dyskinesia score (sum of updrs part iv items 3234), fluctuation score (sum of updrs part iv items 3639), h&y staging and schwab and england activities of daily living (adl);motor subtypes: postural instability gait difficulty (pigd) score (sum of updrs part iii items concerning rise, gait and postural instability) and freezing speech swallowing (foss) score (sum of updrs part ii items on freezing, speech and swallowing);predominance of core manifestations: proportion of updrs part iii on motor scores accounted for tremor (items 2021), rigidity (item 22), bradykinesia (items 2326 and 31) and gait (items 2730) in percentage;asymmetry index: absolute differences in updrs between sides divided by the total updrs part iii items 2026;axial / limb ratio: sum of updrs part iii items 18, 19, 22 and 2730 divided by the sum of updrs part iii items 2026;other motor symptoms: presence of falls and freezing andnonmotor manifestations: the hospital anxiety and depression scale (hads) questionnaire to measure anxiety and depression,27 fatigue severity scale (fss) to investigate fatigue,28 the mini - nutritional assessment (mna) questionnaire together with anthropometric measurements for nutritional status,29 persian - translated and validated version of the scales for outcomes in parkinson s disease psychosocial (scopa - ps) questionnaire30 to assess psychosocial functioning and validated persian version of the 39-item pd questionnaire (pdq-39)31 to evaluate health - related quality of life (hrqol). All clinical assessments were done when the patients were in the on state . Motor severity: unified parkinson s disease rating scale (updrs) subscales i iv, dyskinesia score (sum of updrs part iv items 3234), fluctuation score (sum of updrs part iv items 3639), h&y staging and schwab and england activities of daily living (adl); motor subtypes: postural instability part iii items concerning rise, gait and postural instability) and freezing speech swallowing (foss) score (sum of updrs part ii items on freezing, speech and swallowing); predominance of core manifestations: proportion of updrs part iii on motor scores accounted for tremor (items 2021), rigidity (item 22), bradykinesia (items 2326 and 31) and gait (items 2730) in percentage; asymmetry index: absolute differences in updrs between sides divided by the total updrs part iii items 18, 19, 22 and 2730 divided by the sum of updrs part iii items 2026; other motor symptoms: presence of falls and freezing and nonmotor manifestations: the hospital anxiety and depression scale (hads) questionnaire to measure anxiety and depression,27 fatigue severity scale (fss) to investigate fatigue,28 the mini - nutritional assessment (mna) questionnaire together with anthropometric measurements for nutritional status,29 persian - translated and validated version of the scales for outcomes in parkinson s disease psychosocial (scopa - ps) questionnaire30 to assess psychosocial functioning and validated persian version of the 39-item pd questionnaire (pdq-39)31 to evaluate health - related quality of life (hrqol). All clinical assessments were done when the patients were in the on state . Statistical descriptions and univariate and multivariate regression analyses were performed using the ibm spss statistics for windows, version 23 (ibm corporation, armonk, ny, usa). Mean (standard deviation [sd]) and frequency percentages were used to describe numerical and categorical variables, respectively . Smirnov test was applied to check the normality assumption for continuous variables . Since the normality of distribution was met, we used parametric tests for between - group comparisons . Univariate comparisons between males and females were performed using either independent samples t - test, chi - square test or fisher s exact test where appropriate . We used multivariate linear regression model to adjust the between - group differences in some numeric variables of interest for the baseline differences in disease duration and level of education . A two - tailed p - value of <0.05 was considered as the threshold to show statistical significant differences . In order to evaluate the strength of each variable to discriminate male and female patients with pd, we applied orthogonal partial least squares discriminant analysis (opls - da) method using simca software, version 14.1 (mks umetrics ab, ume, sweden). Unit variance (uv) scaling was used to transform crude data prior to the opls - da modeling . The opls - da method divides the systematic variation in the x - block consisting of a comprehensive list of demographic and pd - related features to separate males and females with pd into two model parts . One part models the co - variation between x and y, and another part expresses the x - variation that is not related to y and is shown by the orthogonal component(s). This method results in a better class resolution for a discriminant problem such as the case in our study . Furthermore, the opls - da method made it possible to compare the strength of different variables with various measurement units and scales to discriminate males and females with pd by means of values of standardized loading, their standard error (se) and 95% confidence interval (ci). We visualized findings from the opls - da method by three different plots with the following parameters: score scatter plot: to show the performance of the entire opls - da model to separate males and females where t1 refers to the score of each participant from the main discriminant component in the x - block (horizontal axis), while t2 shows the score of each participant from the y - orthogonal component (vertical axis);loadings bars: this is a one - dimensional plot representing loading value (p1) of each feature to discriminate males and females based on the main discriminant component of the opls - da model;loadings scatter plot: in this plot, p1 refers to the loading values of each feature from the main discriminant component to discriminate males and females (horizontal axis) and p2 represents the loadings of each variable from the y - orthogonal component for the differences between features that are not related to sex (vertical axis). Score scatter plot: to show the performance of the entire opls - da model to separate males and females where t1 refers to the score of each participant from the main discriminant component in the x - block (horizontal axis), while t2 shows the score of each participant from the y - orthogonal component (vertical axis); loadings bars: this is a one - dimensional plot representing loading value (p1) of each feature to discriminate males and females based on the main discriminant component of the opls - da model; loadings scatter plot: in this plot, p1 refers to the loading values of each feature from the main discriminant component to discriminate males and females (horizontal axis) and p2 represents the loadings of each variable from the y - orthogonal component for the differences between features that are not related to sex (vertical axis). This study was conducted on 157 consecutive patients with ipd from an outpatient referral movement disorders clinic in tehran, iran, between october 2011 and december 2012 . This was a collaborative project between iran university of medical sciences (tehran, iran) and karolinska institute (stockholm, sweden). The study protocol was approved by the ethics committee of the neurology department at firoozgar clinical research development center (fcrdc; affiliated to iran university of medical sciences). Prior to the launch of the study, all patients were informed about the aims and procedures . All participants provided their verbal informed consent to participate in this study . Since the project was designed as an observational research, verbal form of consent was approved by the aforementioned ethics committee . Participation in this study was voluntary, and the patients were free to withdraw from the project whenever they decided . Furthermore, the identity of research participants was protected, since the data files were anonymous and all names were omitted . Recruited patients fulfilled the following inclusion criteria: diagnosis of ipd based on the uk brain bank criteria,25 which was assessed by the same neurologist who was specialized in movement disorders for all participants; current age of 30 years or older and motor disability in the mild - to - severe range but not in the advanced stages that needs wheelchair or hospitalization according to the hoehn and yahr (h&y) criteria (stage <5).26 patients with moderate to severe dementia were excluded from the study, as were those with atypical parkinsonism, including multiple system atrophy (msa), progressive supranuclear palsy (psp) and vascular or drug - induced parkinsonism . Data collection was performed through face - to - face interviews with eligible patients by a trained group of medical interns by means of validated questionnaires and checklists . Patients were also examined for clinical assessments and diagnosis by one neurologist specialized in movement disorders . The demographic checklist consisted of baseline variables (age and sex), educational status, history of smoking, comorbidities, duration of pd (time passed from diagnosis) and history of levodopa administration . Total comorbidity burden was calculated by summing up the number of chronic comorbid conditions in each participant, including depression, hypertension, interstitial heart disease (ihd), diabetes, stroke / transient ischemic attack (tia) and osteoporosis . Data were collected based on participants self - reports and their medical records at the referral center . Clinical characteristics of pd were assessed using the following scales and/or definitions: motor severity: unified parkinson s disease rating scale (updrs) subscales i iv, dyskinesia score (sum of updrs part iv items 3234), fluctuation score (sum of updrs part iv items 3639), h&y staging and schwab and england activities of daily living (adl);motor subtypes: postural instability gait difficulty (pigd) score (sum of updrs part iii items concerning rise, gait and postural instability) and freezing speech swallowing (foss) score (sum of updrs part ii items on freezing, speech and swallowing);predominance of core manifestations: proportion of updrs part iii on motor scores accounted for tremor (items 2021), rigidity (item 22), bradykinesia (items 2326 and 31) and gait (items 2730) in percentage;asymmetry index: absolute differences in updrs between sides divided by the total updrs part iii items 2026;axial / limb ratio: sum of updrs part iii items 18, 19, 22 and 2730 divided by the sum of updrs part iii items 2026;other motor symptoms: presence of falls and freezing andnonmotor manifestations: the hospital anxiety and depression scale (hads) questionnaire to measure anxiety and depression,27 fatigue severity scale (fss) to investigate fatigue,28 the mini - nutritional assessment (mna) questionnaire together with anthropometric measurements for nutritional status,29 persian - translated and validated version of the scales for outcomes in parkinson s disease psychosocial (scopa - ps) questionnaire30 to assess psychosocial functioning and validated persian version of the 39-item pd questionnaire (pdq-39)31 to evaluate health - related quality of life (hrqol). All clinical assessments were done when the patients were in the on state . Motor severity: unified parkinson s disease rating scale (updrs) subscales i iv, dyskinesia score (sum of updrs part iv items 3234), fluctuation score (sum of updrs part iv items 3639), h&y staging and schwab and england activities of daily living (adl); motor subtypes: postural instability part iii items concerning rise, gait and postural instability) and freezing speech swallowing (foss) score (sum of updrs part ii items on freezing, speech and swallowing); predominance of core manifestations: proportion of updrs part iii on motor scores accounted for tremor (items 2021), rigidity (item 22), bradykinesia (items 2326 and 31) and gait (items 2730) in percentage; asymmetry index: absolute differences in updrs between sides divided by the total updrs part iii items 2026; axial / limb ratio: sum of updrs part iii items 18, 19, 22 and 2730 divided by the sum of updrs part iii items 2026; other motor symptoms: presence of falls and freezing and nonmotor manifestations: the hospital anxiety and depression scale (hads) questionnaire to measure anxiety and depression,27 fatigue severity scale (fss) to investigate fatigue,28 the mini - nutritional assessment (mna) questionnaire together with anthropometric measurements for nutritional status,29 persian - translated and validated version of the scales for outcomes in parkinson s disease psychosocial (scopa - ps) questionnaire30 to assess psychosocial functioning and validated persian version of the 39-item pd questionnaire (pdq-39)31 to evaluate health - related quality of life (hrqol). All clinical assessments were done when the patients were in the on state . Statistical descriptions and univariate and multivariate regression analyses were performed using the ibm spss statistics for windows, version 23 (ibm corporation, armonk, ny, usa). Mean (standard deviation [sd]) and frequency percentages were used to describe numerical and categorical variables, respectively . Smirnov test was applied to check the normality assumption for continuous variables . Since the normality of distribution was met, we used parametric tests for between - group comparisons . Univariate comparisons between males and females were performed using either independent samples t - test, chi - square test or fisher s exact test where appropriate . We used multivariate linear regression model to adjust the between - group differences in some numeric variables of interest for the baseline differences in disease duration and level of education . A two - tailed p - value of <0.05 was considered as the threshold to show statistical significant differences . In order to evaluate the strength of each variable to discriminate male and female patients with pd, we applied orthogonal partial least squares discriminant analysis (opls - da) method using simca software, version 14.1 (mks umetrics ab, ume, sweden). Unit variance (uv) scaling was used to transform crude data prior to the opls - da modeling . The opls - da method divides the systematic variation in the x - block consisting of a comprehensive list of demographic and pd - related features to separate males and females with pd into two model parts . One part models the co - variation between x and y, and another part expresses the x - variation that is not related to y and is shown by the orthogonal component(s). This method results in a better class resolution for a discriminant problem such as the case in our study . Furthermore, the opls - da method made it possible to compare the strength of different variables with various measurement units and scales to discriminate males and females with pd by means of values of standardized loading, their standard error (se) and 95% confidence interval (ci). We visualized findings from the opls - da method by three different plots with the following parameters: score scatter plot: to show the performance of the entire opls - da model to separate males and females where t1 refers to the score of each participant from the main discriminant component in the x - block (horizontal axis), while t2 shows the score of each participant from the y - orthogonal component (vertical axis);loadings bars: this is a one - dimensional plot representing loading value (p1) of each feature to discriminate males and females based on the main discriminant component of the opls - da model;loadings scatter plot: in this plot, p1 refers to the loading values of each feature from the main discriminant component to discriminate males and females (horizontal axis) and p2 represents the loadings of each variable from the y - orthogonal component for the differences between features that are not related to sex (vertical axis). Score scatter plot: to show the performance of the entire opls - da model to separate males and females where t1 refers to the score of each participant from the main discriminant component in the x - block (horizontal axis), while t2 shows the score of each participant from the y - orthogonal component (vertical axis); loadings bars: this is a one - dimensional plot representing loading value (p1) of each feature to discriminate males and females based on the main discriminant component of the opls - da model; loadings scatter plot: in this plot, p1 refers to the loading values of each feature from the main discriminant component to discriminate males and females (horizontal axis) and p2 represents the loadings of each variable from the y - orthogonal component for the differences between features that are not related to sex (vertical axis). Overall, 157 individuals consisting of 108 males and 49 females with pd were recruited in our study . Table 1 summarizes the results for univariate comparison of the demography, motor severity and medications, motor subtypes, nonmotor features and qol indicators between the two subgroups . The age at pd onset did not significantly differ between males and females (55.2 [sd = 11.6] year vs 53.4 [sd = 12.4] year, p=0.384), however, disease duration was longer in females at the time of recruitment (6.1 [sd = 4.9] year vs 8.2 [sd = 5.7] year, p=0.023). Univariate differences in motor features such as total updrs and freezing score disappeared after adjustment for baseline difference . Among nonmotor features, anxiety, depression, fatigue and psychosocial functioning were all significantly more severe in female pd patients (table 1). After statistical adjustment for the baseline differences in disease duration and level of education, female individuals still showed significantly higher score in anxiety (mean difference = 2.2 [95% ci: 0.54.0], p=0.011) and borderline higher score in depression (mean difference = 1.3 [95% ci: 0.22.7], p=0.079). Furthermore, female pd patients had significantly worse nutritional status and higher parkinson s disease summary index (pdsi), both of which remained statistically significant after multivariate adjustment (mean difference for mna score = 1.5 [95% ci: 2.5 to 0.4], p=0.009; mean difference for pdsi score = 6.9 [95% ci: 1.911.8], p=0.006). Among different domains of qol, females were significantly worse in mobility (40.8 [sd = 27.4] vs 22.5 [sd = 24.0], p<0.001), emotional well - being (39.4 [sd = 24.4] vs 23.5 [sd = 21.7], p<0.001), social support (20.1 [sd = 25.9] vs 7.5 [sd = 15.9], p<0.001) and bodily discomfort (33.0 [sd = 25.3] vs 17.2 [sd = 20.1], p<0.001). All of these differences were still significant even after multivariate adjustment for the baseline differences in disease duration and level of education between males and females, resulting in the mean difference of 11.9 (95% ci: 3.520.3, p=0.006) in mobility, 12.5 (95% ci: 4.720.3, p=0.002) in emotional well - being and 11.0 (95% ci: 0.921.5, p=0.001) and 14.2 (95% ci: 6.421.9, p<0.001) in bodily discomfort domain of qol . The opls - da model that fitted the best to our dataset had the overall cross - validated predictive r value of 0.33 . The score scatter plot in figure 1 shows the performance of this model to discriminate males and females with pd . The corresponding loading value of each variable in the opls - da model and its 95% ci are shown in figure 2, and the loadings scatter plot is illustrated in figure 3 . Based on the absolute loading value for each variable in the opls - da model, emotional well - being (mean = 0.22, se = 0.14), bodily discomfort (mean = 0.18, se = 0.13), social support (mean = 0.17, se = 0.19), mobility (mean = 0.17, se = 0.04) and communication (mean = 0.12, se = 0.15) domains of qol, together with anxiety (mean = 0.18, se = 0.10), depression (mean = 0.17, se = 0.08) and psychosocial functioning (mean = 0.16, se = 0.11), were the strongest features with higher values in favor of females to discriminate the two sexes in pd population (figures 2 and 3). Nutritional status (mean = 0.13, se = 0.07), schwab and england adl score (mean = 0.06, se = 0.13), and orthostasis (mean = 0.06, se = 0.05) were also found to be strong discriminators in the model, nevertheless, with higher values in favor of the male pd patients . The age at pd onset did not significantly differ between males and females (55.2 [sd = 11.6] year vs 53.4 [sd = 12.4] year, p=0.384), however, disease duration was longer in females at the time of recruitment (6.1 [sd = 4.9] year vs 8.2 [sd = 5.7] year, p=0.023). Univariate differences in motor features such as total updrs and freezing score disappeared after adjustment for baseline difference . Among nonmotor features, anxiety, depression, fatigue and psychosocial functioning were all significantly more severe in female pd patients (table 1). After statistical adjustment for the baseline differences in disease duration and level of education, female individuals still showed significantly higher score in anxiety (mean difference = 2.2 [95% ci: 0.54.0], p=0.011) and borderline higher score in depression (mean difference = 1.3 [95% ci: 0.22.7], p=0.079). Furthermore, female pd patients had significantly worse nutritional status and higher parkinson s disease summary index (pdsi), both of which remained statistically significant after multivariate adjustment (mean difference for mna score = 1.5 [95% ci: 2.5 to 0.4], p=0.009; mean difference for pdsi score = 6.9 [95% ci: 1.911.8], p=0.006). Among different domains of qol, females were significantly worse in mobility (40.8 [sd = 27.4] vs 22.5 [sd = 24.0], p<0.001), emotional well - being (39.4 [sd = 24.4] vs 23.5 [sd = 21.7], p<0.001), social support (20.1 [sd = 25.9] vs 7.5 [sd = 15.9], p<0.001) and bodily discomfort (33.0 [sd = 25.3] vs 17.2 [sd = 20.1], p<0.001). All of these differences were still significant even after multivariate adjustment for the baseline differences in disease duration and level of education between males and females, resulting in the mean difference of 11.9 (95% ci: 3.520.3, p=0.006) in mobility, 12.5 (95% ci: 4.720.3, p=0.002) in emotional well - being and 11.0 (95% ci: 0.921.5, p=0.001) and 14.2 (95% ci: 6.421.9, p<0.001) in bodily discomfort domain of qol . The opls - da model that fitted the best to our dataset had the overall cross - validated predictive r value of 0.33 . The score scatter plot in figure 1 shows the performance of this model to discriminate males and females with pd . The corresponding loading value of each variable in the opls - da model and its 95% ci are shown in figure 2, and the loadings scatter plot is illustrated in figure 3 . Based on the absolute loading value for each variable in the opls - da model, emotional well - being (mean = 0.22, se = 0.14), bodily discomfort (mean = 0.18, se = 0.13), social support (mean = 0.17, se = 0.19), mobility (mean = 0.17, se = 0.04) and communication (mean = 0.12, se = 0.15) domains of qol, together with anxiety (mean = 0.18, se = 0.10), depression (mean = 0.17, se = 0.08) and psychosocial functioning (mean = 0.16, se = 0.11), were the strongest features with higher values in favor of females to discriminate the two sexes in pd population (figures 2 and 3). Nutritional status (mean = 0.13, se = 0.07), schwab and england adl score (mean = 0.06, se = 0.13), and orthostasis (mean = 0.06, se = 0.05) were also found to be strong discriminators in the model, nevertheless, with higher values in favor of the male pd patients . Our study is one of the few attempts to comprehensively investigate sexual dimorphism in pd . We applied powerful multivariate statistical methods to explore independent sex differences in clinical manifestations, hrqol and psychosocial functioning of people with pd . In general, females with pd were significantly worse in psychological features such as anxiety and depression, nutritional status and specific domains of qol, namely, mobility, emotional well - being, social support and bodily discomfort . Studies have suggested that emotional symptoms of pd, especially depression, are more common and more severe in females than in males.3234 however, these findings were not approved by a large prospective study, which found no difference in the prevalence or severity of depression evaluated by beck depression inventory (bdi) between females and males.24 in our study, females with pd tended to be more affected from psychological symptoms . We found that the scores for anxiety, psychosocial functioning and emotional well - being were all higher among females demonstrating a worse status . Conflicting results can be, in part, explained by ethnic, cultural and environmental differences as well as the use of different depression scales . In some cultures and environments, women are more prone to be stigmatized by disabling conditions,35,36 which make them more vulnerable to emotional and psychosocial adverse effects of pd . Social and physical well - being, measured separately or as parts of qol questionnaires, are important contributors of patient s qol.37,38 in our study, physical discomfort, including mobility, fatigue and bodily discomfort, was more severe among female participants . This finding is confirmed by previous studies of pd populations,17,34,39 as well as those of general population showing more severe physical discomfort in healthy older women compared to males of the same age group.4042 our results showed that despite the lack of statistically significant difference in social stigma, females displayed worse communication and psychosocial functioning . These sex - related differences can be the consequences of more severe emotional disturbance, physical discomfort, and poorer social supports in females compared to males with pd . In our study, self - reported cognitive impairment was higher in female participants based on the cognition dimension of pdq-39 . In contrast, some studies have suggested that cognitive decline in pd is more severe among males.18,23 nonetheless, as different dimensions of cognitive performance have been shown to be sex related in healthy elderly adults (for instance, males have better visuospatial ability, while females perform better on face and verbal recognition and semantic fluency tests4345), different dimensions of cognitive performance must be investigated in order to clarify sexual dimorphism of cognition in pd . Tremor and dyskinesia are reported to occur more frequently in females,8,18,33 whereas gait disturbance is more common in males.19 however, in line to one prospective study that had also adjusted the differences for the confounding effects of demographics and clinical covariates,24 we also could not find sex - related differences in motor symptoms . Sleep quality is the other symptom of pd, which is believed to be sex related, but still remained a controversial issue . Rapid eye movement (rem) sleep behavior disorder (rbd) was reported as a male - related symptom in pd;33,4648 some other sleep disturbances were found to be more severe among females.34,49 we did not find any difference in sleep disturbance between males and females using the nonmotor items of the updrs . A more thorough assessment with a valid and specific tool such as polysomnography is required to investigate sexual dimorphism in sleep disorders in people with pd . The comprehensive list of pd - related features and general aspects of daily life is the most important strength of our study . Previous studies have investigated sexual dimorphism in manifestations of pd with rather small sample size not being controlled for the potential confounders.3,50 in our study, we evaluated a large list of variables (some of which were considered for sexual dimorphism in pd for the first time) and applied a strong multivariate statistical approach, opls - da, to take into account all potential confounding interactions this study was designed as a cross - sectional research that restricts any causal inferences and going beyond associations . There are other aspects of sexual dimorphism in pd that need to be addressed in future research, namely, course of progression and mortality . There is a potential risk for selection bias since all recruited patients were from an outpatient neurology clinic where the majority of patients had mild - to - moderate ipd . As a result, our findings may not be generalized to all people with pd, particularly those with end - stage disease . Considering time restriction for data collection spent in each participant, we ought to rely on the items from updrs for some variables, which might not be sensitive enough to show between - group differences on this summary scale . Therefore, more sensitive gold - standard tools might potentially show different results for such features . Our rather small sample size in comparison with few large previous studies should also be noted . Augustine et al24 reported that studies with smaller sample sizes were more likely to report sex differences in different clinical features . We should also add the uneven number of males and females in our study as another limitation; however, it could be expected for pd that affects men more frequently.51 yet the comprehensive list of variables and appropriate multivariate statistical methods of the present study overcame these issues and strengthened our findings . This study provides a comprehensive understanding of sexual dimorphism in different motor and nonmotor features of pd and various domains of daily life by using sophisticated statistical analysis to evaluate the independent differences between males and females . After controlling for potential confounders, anxiety, depression, mobility, emotional well - being, social support, on the other hand, male pd patients had better nutritional status (though with rather small effect size for difference) and adl but also more severe orthostasis . Conclusively concordance of depression, anxiety and overall psychosocial burden in being more severe in women is in favor of the presence of a considerable sex dimorphism in neuropsychiatric symptoms, which in turn suggest its clinical importance . Qol and psychosocial consequences of pd between males and females should be addressed for developing a more personalized caring approach . These aspects of sexual dimorphism in pd also enlighten the features that are more likely to be affected in each sex and should be specifically targeted when managing male and female individuals with pd . As for clinical implication, clinicians should consider sex differences in the evaluation and management of patients with pd . For example, they should be aware of the higher risk of psychological manifestations in women and therefore should screen them accurately in a timely manner and, if necessary, refer them to a psychologist in the early stages of these manifestations . Sex - specific symptoms highlight the importance of sex - specific treatments; hence, future studies should investigate the effects of specific interventions for females . Moreover, educational interventions are also needed in order to improve social support for women with pd and reduce their extra psychosocial burden.
Due to its dismal prognosis, pancreatic cancer is the fourth most common cause of cancer death in europe and the usa . From a clinical point of view therefore, complete surgical resection of the primary tumor represents the only curative treatment option . However, even patients with tumor - free margins (r0 resection) experience frequently local recurrence and distant metastases, which is in clear contrast to other solid tumors of the gastrointestinal tract . Consequently, more radical approaches have been evaluated, first described by fortner and colleagues . Even though there was initial indication of some survival benefit, this raised the question as to whether such a discrepancy is caused, other than through incomplete lymphadenectomy and perineural invasion, by a misclassification of r1 resections as r0 resections . According to the recent literature, the rates of noncurative resections range from 15% to 35% [12, 15, 23, 27], whereas postmortem examinations of pancreatic cancer patients revealed local recurrence rates approaching 100% [5, 7, 19, 21, 24]. This is supported by recent publications (verbeke et al . And esposito et al .) Revealing that modified histopathological workup of pancreatic head carcinomas leads to r1 resection rates of 85% and 76%, respectively . The aim of the present article was to develop an optimized and standardized histopathological workup and to test prospectively the hypothesis that current histopathological reports underestimate the proportion of r1 pancreatic head resections . Towards this goal, we firstly implemented color coding of the resection margins (rms) and the organ surfaces . Secondly, we carefully reevaluated the different sites of r1 resections according to the color code and demonstrated that the mesopancreatic rm was the most frequent site of incomplete tumor resection in order to test the hypothesis that an optimized and standardized histopathological workup increases the rate of r1 resections after pancreatic head resections, we first compared the rates of curative resections after conventional histopathological workup in our department . Therefore, we retrospectively identified all patients who had undergone pancreatic head resection, either through pylorus - preserving pancreaticoduodenectomy (pppd) or kausch whipple procedure due to malignant diseases, in the department of general and visceral surgery between 1996 and 2005 . During this period, the location, histological tumor type, size of tumor, and lymph node involvement were defined . Assessment of the rm included the common bile duct, the pancreatic transection margin, the duodenal and jejunal resection plane, and the anterior and posterior surface . Cases with macroscopic tumor residues at the surgical rm or the organ surface were defined as r2 . If tumor cells at the surgical rm or the organ surface were only detectable microscopically, the resection was classified r1 . A curative r0 resection was defined as a surgical rm or organ surface without tumor cell infiltration . A modified and standardized histopathological workup was introduced in april 2006 and prospectively tested for 28 months until july 2008 . Organ surfaces and rms of the pancreatic head resection specimen were stained according to a well - defined five - color code (fig . 1): the anterior (ventral) surface was painted black, the posterior (dorsal) surface white, the groove of the superior mesenteric vein (smv) green, the pancreatic transection margin yellow, and the mesopancreas red . The mesopancreas was defined as the soft tissue between the superior mesenteric artery and the pancreatic parenchyma and contains lymphatic, nervous, and vascular structures . Since its identification is challenging, especially after formalin fixation, the mesopancreas was stained directly after removing the specimen from the situs, while the other parts were colored after formalin fixation for 24 to 36 h. all staining procedures were performed by the operating surgeon or by a surgeon present during the procedure . 1a d stained surfaces of pancreatic head resection specimen (black ventral; white dorsal; yellow pancreatic rm; green groove of superior mesenteric vein; red mesopancreatic rm) a d stained surfaces of pancreatic head resection specimen (black ventral; white dorsal; yellow pancreatic rm; green groove of superior mesenteric vein; red mesopancreatic rm) after fixation and staining, the rms of the proximal duodenum / stomach, distal duodenum, the common bile duct, and if present any major vessel (smv, portal vein) were identified and completely embedded . The specimen was serially sliced (0.5- to 1-cm slices) perpendicular to the mesopancreatic rm (fig . Several samples were taken from the tumor with relation to the anterior and posterior surface . The distance between the tumor and each colored surface or resection margin was measured microscopically and documented in the pathohistological report . Metastasis) was carried out according to the current world health organization and international union against cancer (uicc) criteria . According to the uicc criteria, the operation was considered as potentially curative (r0) if the rms and organ surfaces were free of tumor cells, whereas histopathologically verified tumor cell infiltration was defined as r1 resection . In cases of macroscopically visible tumor tissue, the resection was classified r2 . Applying the definition of the royal college of pathologists (rcp), the specimens were classified r1 if tumor cells were within 1 mm of the rm . Histopathological inclusion criteria were diagnosis of either pancreatic ductal adenocarcinoma (pdac), distal bile duct adenocarcinomas (dbd), or periampullary adenocarcinoma (pac). Ac indicates the adenocarcinoma, mp the mesopancreas section planes indicated at resection specimen (a). Individual slices (b) and magnification of one representative slice (c). Ac indicates the adenocarcinoma, mp the mesopancreas the vast majority of pancreatic head resections were performed as pppd . All resection procedures were performed by two experienced surgeons (bmg, hb). Since an extended lymphadenectomy does not necessarily improve patient outcome, we rather intended to achieve oncological radicality through the extended excision of perivascular tissue around the superior mesenteric artery (sma). To achieve this goal, the sma was approached caudal to the region of the uncinate process, where a distinct fascia separates the uncinate from the mesocolon . Then, the sma was isolated laterally right up to the vessel, and all tissues between the artery and the pancreatic parenchyma were resected . Attention was paid not to dissect it entirely from the surrounding mesenteric plexus to avoid postoperative diarrhea (see fig . 3). Lymph node dissection was performed up to the hepatoduodenal ligament laterally to the portal vein . Where there was evidence of tumor infiltration, the smv was partially resected and reconstructed either by end - to - end anastomosis or insertion of a venous graft . Finally, the posterior surface of the pancreatic head specimen was assessed macroscopically for its integrity . 3intraoperative view before (left) and after resection of the mesopancreas (right). Small arrows point to the superior mesenteric artery; large arrow indicates the resection line; dashed line indicates dissection plane (left; smv superior mesenteric vein; sma superior mesenteric artery; pv portal vein) intraoperative view before (left) and after resection of the mesopancreas (right). Small arrows point to the superior mesenteric artery; large arrow indicates the resection line; dashed line indicates dissection plane (left; smv superior mesenteric vein; sma superior mesenteric artery; pv portal vein) in order to test the hypothesis that an optimized and standardized histopathological workup increases the rate of r1 resections after pancreatic head resections, we first compared the rates of curative resections after conventional histopathological workup in our department . Therefore, we retrospectively identified all patients who had undergone pancreatic head resection, either through pylorus - preserving pancreaticoduodenectomy (pppd) or kausch whipple procedure due to malignant diseases, in the department of general and visceral surgery between 1996 and 2005 . During this period, the location, histological tumor type, size of tumor, and lymph node involvement were defined . Assessment of the rm included the common bile duct, the pancreatic transection margin, the duodenal and jejunal resection plane, and the anterior and posterior surface . Cases with macroscopic tumor residues at the surgical rm or the organ surface were defined as r2 . If tumor cells at the surgical rm or the organ surface were only detectable microscopically, the resection was classified r1 . A curative r0 resection was defined as a surgical rm or organ surface without tumor cell infiltration . A modified and standardized histopathological workup was introduced in april 2006 and prospectively tested for 28 months until july 2008 . Organ surfaces and rms of the pancreatic head resection specimen were stained according to a well - defined five - color code (fig . 1): the anterior (ventral) surface was painted black, the posterior (dorsal) surface white, the groove of the superior mesenteric vein (smv) green, the pancreatic transection margin yellow, and the mesopancreas red . The mesopancreas was defined as the soft tissue between the superior mesenteric artery and the pancreatic parenchyma and contains lymphatic, nervous, and vascular structures . Since its identification is challenging, especially after formalin fixation, the mesopancreas was stained directly after removing the specimen from the situs, while the other parts were colored after formalin fixation for 24 to 36 h. all staining procedures were performed by the operating surgeon or by a surgeon present during the procedure . 1a d stained surfaces of pancreatic head resection specimen (black ventral; white dorsal; yellow pancreatic rm; green groove of superior mesenteric vein; red mesopancreatic rm) a d stained surfaces of pancreatic head resection specimen (black ventral; white dorsal; yellow pancreatic rm; green groove of superior mesenteric vein; red mesopancreatic rm) after fixation and staining, the rms of the proximal duodenum / stomach, distal duodenum, the common bile duct, and if present any major vessel (smv, portal vein) were identified and completely embedded . The specimen was serially sliced (0.5- to 1-cm slices) perpendicular to the mesopancreatic rm (fig . Several samples were taken from the tumor with relation to the anterior and posterior surface . The distance between the tumor and each colored surface or resection margin was measured microscopically and documented in the pathohistological report . Metastasis) was carried out according to the current world health organization and international union against cancer (uicc) criteria . According to the uicc criteria, the operation was considered as potentially curative (r0) if the rms and organ surfaces were free of tumor cells, whereas histopathologically verified tumor cell infiltration was defined as r1 resection . In cases of macroscopically visible tumor tissue, the resection was classified r2 . Applying the definition of the royal college of pathologists (rcp), the specimens were classified r1 if tumor cells were within 1 mm of the rm . Histopathological inclusion criteria were diagnosis of either pancreatic ductal adenocarcinoma (pdac), distal bile duct adenocarcinomas (dbd), or periampullary adenocarcinoma (pac). Ac indicates the adenocarcinoma, mp the mesopancreas section planes indicated at resection specimen (a). Individual slices (b) and magnification of one representative slice (c). All resection procedures were performed by two experienced surgeons (bmg, hb). Since an extended lymphadenectomy does not necessarily improve patient outcome, we rather intended to achieve oncological radicality through the extended excision of perivascular tissue around the superior mesenteric artery (sma). To achieve this goal, the sma was approached caudal to the region of the uncinate process, where a distinct fascia separates the uncinate from the mesocolon . Then, the sma was isolated laterally right up to the vessel, and all tissues between the artery and the pancreatic parenchyma were resected . Attention was paid not to dissect it entirely from the surrounding mesenteric plexus to avoid postoperative diarrhea (see fig . Lymph node dissection was performed up to the hepatoduodenal ligament laterally to the portal vein . Where there was evidence of tumor infiltration, the smv was partially resected and reconstructed either by end - to - end anastomosis or insertion of a venous graft . Finally, the posterior surface of the pancreatic head specimen was assessed macroscopically for its integrity . 3intraoperative view before (left) and after resection of the mesopancreas (right). Small arrows point to the superior mesenteric artery; large arrow indicates the resection line; dashed line indicates dissection plane (left; smv superior mesenteric vein; sma superior mesenteric artery; pv portal vein) intraoperative view before (left) and after resection of the mesopancreas (right). Small arrows point to the superior mesenteric artery; large arrow indicates the resection line; dashed line indicates dissection plane (left; smv superior mesenteric vein; sma superior mesenteric artery; pv portal vein) in order to compare the rates of curative and noncurative resections in our department to the published data, we retrospectively identified 115 patients with malignant pancreatic head tumors who had undergone either pppd or a kausch whipple procedure between 1996 and 2005 (n = 115). For 89 of these 115 patients (77.4%), the operation could be considered potentially curative, whereas the rms of 26 cancer specimens were positive (22.6%). In april 2006, we introduced the modified and standardized histopathological workup and applied it prospectively until july 2008 . During this 28-month period, whipple procedure: three patients as a consequence of advanced disease, one owing to inflammatory changes of the tissue, and one owing to the intraoperative detection of a synchronous gastric neoplasia . None of the 100 patients died within 30 days after surgery . A 79-year - old patient who underwent head resection for ductal adenocarcinoma developed endocarditis with consecutive mitral valve regurgitation . He required replacement of the mitral valve but died postoperatively as a result of the cardiac procedure . After definitive histopathological assessment, 35 patients were excluded from this analysis, owing to histopathological diagnosis as listed in table 1 . Table 1pancreatic head specimens excluded from analysisno.nonmalignant or borderlineno.malignant7chronic pancreatitis5neuroendocrine tumor4autoimmune pancreatitis4metastases to the pancreatic head2periampullary adenoma1duodenal adenocarcinoma2periampullary adenomyoma1acinar cell carcinoma2serous cystadenoma1non - hodgkin lymphoma2intraductal papillary mucinous neoplasia (ipmn)1mucinous cystadenoma1duodenitis1simple pancreatic cyst1duodenal diverticulum23total12total pancreatic head specimens excluded from analysis therefore, 65 pancreatic head resections with a malignant tumor were analyzed further (pac, n = 7; dbd, n = 12; pdac, n = 46). Applying the uicc criteria, 32 cancers were curatively (r0) resected (49.2%), while 33 cases turned out to be r1 resections (50.8%; table 2). Interestingly, the mesopancreas was the only site of infiltration in 17 of these r1 specimens (51.5%). In another five cases, infiltration of the mesopancreas with additional involvement of the smv or the pancreatic transection margin was discovered . In all, the mesopancreas was determined as being the most frequent site with residual tumor mass by far (n = 22, 56.4%). The groove of the smv (n = 1), the anterior (n = 2) and posterior surface (n = 1), the pancreatic transection margin (n = 4), and the proximal duodenum (n = 2) were only infiltrated infrequently in the final histological diagnosis . Smv margins were infiltrated in seven of the 17 specimens including partial or complete smv resection . Applying the definitions of the rcp, an additional set of 13 specimens would have to be considered as r1 resections, resulting in a total percentage of 70.8% of noncurative operations . As expected, the total number of affected areas increased from 39 to 83, involving the mesopancreas (n = 27) and the anterior (n = 18) and the posterior (n = 13) surface most often (table 2). Table 2histopathological and resection classification dataall cancerspdacdbdpacuiccrcpuiccrcpuiccrcpuiccrcpresectionr032 (49.2%)19 (29.2%)17 (37.0%)8 (17.4%)10 (83.3%)7 (58.3%)5 (71.4%)4 (57.1%)r1/r233 (50.8%)46 (70.8%)29 (63.0%)38 (82.6%)2 (16.7%)5 (41.7%)2 (28.6%)3 (42.9%)site of r1mesopancreas22 (56.4%)27 (32.5%)19 (57.6%)24 (34.3%)1 (25.0%)1 (11.1%)2 (100%)2 (50.0%)pancreatic transection margin4 (10.3%)11 (13.3%)3 (9.1%)10 (14.3%)1 (25.0%)1 (11.1%)00anterior2 (5.1%)18 (21.7%)2 (6.1%)15 (21.4%)01 (11.1%)02 (50.0%)posterior1 (2.6%)13 (15.7%)1 (3.0%)11 (15.7%)02 (22.2%)00groove of smv1 (2.6%)4 (4.8%)1 (3.0%)2 (2.9%)02 (22.2%)00smv (n = 17)7 (17.9%)8 (9.6%)5 (15.2%)6 (8.6%)2 (50.0%)2 (22.2%)00duodenum oral2 (5.1%)2 (2.4%)2 (6.1%)2 (2.9%)0000number of infiltrated sites126 (78.8%)22 (47.8%)25 (86.2%)18 (47.4%)02 (40%)2 (100%)2 (66.7%)27 (21.2%)14 (30.4%)4 (13.8%)11 (28.9%)2 (100%)2 (40%)01 (33.3%)308 (17.4%)07 (18.4%)01 (20%)00401 (2.2%)01 (2.6%)0000501 (2.2%)01 (2.6%)0000total398333704924t12 (3.1%)2 (4.3%)00t21 (1.5%)001 (14.3%)t350 (76.9%)43 (93.5%)6 (50%)1 (14.3%)t412 (18.5%)1 (2.2%)6 (50%)5 (71.4%)n012 (18.5%)7 (15.2%)4 (33.3%)1 (14.3%)n153 (81.5%)39 (84.8%)8 (66.7%)6 (85.7%)resection of 13 pdac and four dbd included an smv resectionuicc international union against cancer, rcp royal college of pathologists, smv superior mesenteric vein, pdac pancreatic ductal adenocarcinoma, dbd distal bile duct adenocarcinoma, pac periampullary adenocarcinoma histopathological and resection classification data resection of 13 pdac and four dbd included an smv resection uicc international union against cancer, rcp royal college of pathologists, smv superior mesenteric vein, pdac pancreatic ductal adenocarcinoma, dbd distal bile duct adenocarcinoma, pac periampullary adenocarcinoma pdac represent 71% of all analyzed cancer specimens . Looking at each histopathological type, it became obvious that the number of r1 resections is much lower for dbd (16.7% or 41.7% applying uicc or rcp criteria) and pac (28.6% or 42.9%), irrespective of the classification . Although the total number of r1 resected specimens is too small to draw definitive conclusions, it is noteworthy that the mesopancreas is infiltrated in two of the two r1 resected pac . Surgery is still considered to be the only potentially curative approach if complete resection is possible [6, 20, 23]. However, surgical resection in patients with localized pancreatic cancer is still underused . Retrospective analyses of data from patients treated in our department between 1996 and 2005 revealed a 5-year survival rate of 20% for r0 patients, which is in agreement with the literature, even though some authors deny the existence of 5-year survivors . With respect to the classification of the pancreatic head resections evaluated retrospectively, the percentage of r1 resections (22.6%) also mirrors the literature [12, 15, 23, 27]. These data appear to indicate the possible misclassification of r1 resections as r0 . Local recurrence and disseminated cancer this is supported by postmortem examinations reporting a local recurrence rate approaching 100% [5, 7, 19, 21, 24]. [8, 13] already stated in 1999 that tumor recurrence is primarily due to incomplete removal at the site of resection rather than to metastatic disease . For the 54 pancreatic head resections, a color code was implemented to distinguish between the anterior, posterior, and smv groove surface . Similar results were published by esposito et al . Who reported an r1 resection rate of 76% in 111 pdac . As part of their protocol, they color - coded the anterior, posterior, medial margin, and groove surface of the smv, respectively, whereas the retroperitoneal rm was not investigated separately . The importance of the retroperitoneal resection margin was confirmed by westgaard et al . Who thoroughly worked up 114 periampullary adenocarcinomas by perpendicular sectioning . The overall r1 resection rate was 35%, whereas the retroperitoneal resection margin was involved in 80% of the specimens . However, it should be pointed out that all three studies classified the specimens according to the definition of the royal college of pathologists . Since there do not exist any general guidelines about the definition of r1 resection, we primarily applied the uicc criteria, i.e., a curative resection was defined as there being no microscopic evidence of residual tumor at the rm . We found an r1 resection rate of 50.8%, which demonstrates that a high rate of r1 resections is not only based on the definition of margin positivity as tumor clearance of 1 mm . Our r1 rate (using rcp criteria) was comparable to those from verbeke et al . And esposito et al . Therefore, we could confirm that the number of noncurative resections is considerably higher when a modified and intensified histopathological workup is applied . Interestingly, westgaard et al . Only investigated the transection margins of the pancreas and reached r1 rate of 45% for pdac and 59% for dbd . We identified the mesopancreas as the primary site of positive rms . In total, 66.6% of those cancers resected noncuratively displayed infiltration of this structure . As pointed out by other authors [8, 25] some consider the complete posterior surface of the pancreas as retroperitoneal margin, while others use the term to describe just the area of sharp dissection, similar to what we call mesopancreatic rm . . Defined medial and posterior soft tissue margins to acknowledge the importance of the region . Therefore, we introduced the term mesopancreatic rm to describe the dissection margin in the peripancreatic fatty tissue behind the pancreatic head and lateral to the mesenteric artery . The tissue between the pancreatic parenchyma and the sma consists of fatty tissue and contains blood and lymphatic vessels, as well as lymph nodes draining the pancreatic head and the uncinate . Additionally, nerve fibers are located in the mesopancreas innervating the pancreatic parenchyma . In this context, these structures should be defined as mesopancreas although the peritoneal attachment, which is a requisite to the existence of a meso, was lost during embryological development owing to duodenal rotation . The mesopancreas rises from the embryological mesentery attaching the pancreatic bud to the abdominal wall . This leads to the consequence that the mesopancreas must be resected and removed completely in its integrity down to the mesenteric artery . Based on the embryological development and according to our experience, the pancreatic tissue is not directly adjacent to the mesenteric artery but connected by a delicate meshwork of connective tissue rich in nerve fibers (mesenteric plexus). This layer should be separated close to the mesopancreas to prevent the complications of postoperative diarrhea . As verbeke and colleagues demonstrated, intensive embedding of resected tissue increases the rate of r1 resections . However, infiltration of the mesopancreas may have been misinterpreted in the past as a consequence of a cancer - induced fibrosis or disseminated cancer cells . Therefore, rms in their integrity and numerous samples from the tumor with relation to the surfaces of the pancreas were embedded as part of this modified and standardized protocol . It is widely accepted that only an r0 resection is considered as a curative approach and stratification for resection status is routinely performed in clinical trials such as in the conko-001 trial . Numerous trials failed to show significant differences in patients treatment which could possibly be due to the misinterpretation of the resection status based on the lack of a standardized workup . In general, r1 resections rates of about 20% are reported [11, 16, 18, 27]. However, one can speculate that in these trials the low r1 resection rates are due to a conventional pathohistological workup . Being aware of the crucial importance of assessing the resection rate an intensified workup is obligatory in trials such as conko-005 and conko-006 . Considering the mesopancreas as a structure guiding relevant vessels for lymphatic and blood drainage of the pancreatic head and the uncinate, it may be hypothesized that tumor cells drain into the lymphatic and blood vessels during manipulation of the pancreatic head (e.g., kocher s maneuver). As in the treatment of colorectal cancer, a no - touch technique would diminish this potential influence, resulting in the need to resect the mesopancreas along the superior mesenteric artery first . Based on our data, we propose a standardized histopathological workup for pancreatic head cancer specimens, which may represent a more accurate assessment of curative and noncurative resection rates . As a consequence of this modified protocol, we demonstrate that the mesopancreas is a frequent site for positive resection margins, which has potential therapeutic implications and should be considered in clinical trials when stratifying patients into r0 and r1 resection classifications . Owing to morphological changes during formalin fixation, we strongly believe that the complete and meticulous surgical resection of the mesopancreas as the structure to the right of the mesenteric artery must become the standard surgical approach in pancreatic head resection.
Clear - cell sarcoma (ccs) is a rare malignant connective tissue tumor that was first described by enzinger in 1965 . Ccs occurs most commonly in adolescents and young adults and the patients aged> 60 years are rare . It has a high rate of local recurrence, regional lymph node metastasis, and distant metastasis [1, 2]. Ccs occurs most commonly in the deep soft tissues of the extremities, with rare involvement of the head, neck, and trunk . We experienced a very rare case of primary ccs in the mediastinum with successful surgical resection in an elderly patient . In october 2011, a 63-year - old man presented with a mass in the right upper mediastinum on chest radiograph . Computed tomography showed a large oval mass (55 55 85 mm) with clear margins and heterogeneous enhancement in the right upper posterior mediastinum between the phrenic nerve and vertebrae (fig . 1b), and 18f - fluorodeoxyglucose positron emission tomography showed abnormal uptake with a maximum standardized uptake value of 15.1 . The tumor was excised by video - assisted thoracoscopic surgery, with negative surgical margins . The tumor was soft, well circumscribed, encapsulated, and did not invade the pleura . Histological examination of the resected tumor with hematoxylin and eosin (he) staining showed morphological features compatible with conventional soft tissue ccs . The tumor cells were distributed in nests separated by fibrous connective tissue, showing an alveolar pattern (fig . The tumor cells had eosinophilic and clear cytoplasm and oval vesicular nuclei of varying sizes with characteristic prominent eosinophilic nucleoli . 2c), s-100 protein, calretinin, cd34, cd56, cd68, and cd117; and negative for melan - a, cytokeratins ae1ae3, cytokeratin cam5.2, chromogranin a, desmin, caldesmon, myogenin, epithelial membrane antigens, cd57, cd99, and d2 - 40 . Ccs is a very rare soft tissue neoplasm, which occurs in the extremities in 9095% of cases . Our case was diagnosed with a primary ccs in the mediastinum . A search of the english literature revealed only one previously reported case of primary ccs in the mediastinum . Recent cytogenetic studies showed that ccs is associated with the translocation t(12;22) (q13;q12); this translocation is observed in up to 93% of patients with ccs, but never observed in malignant melanoma . In our case, we were unable to determine whether this translocation was present, because of the lack of availability of such analysis in japan . The rarity of ccs in the mediastinum makes it difficult to draw conclusions regarding prognostic factors . Reported that complete excision of the primary tumor with wide surgical margins appears to be the optimal approach to treatment, with or without adjuvant radiation therapy . Ccs is an aggressive malignant tumor, and unlike most soft tissue sarcomas, it often metastasizes to regional lymph nodes . In our case, we did not have a definitive diagnosis at the time of operation, and therefore we did not perform excision of the tumor with wide margins or dissect the regional lymph nodes . In most cases, ccs has a relentlessly progressive course and results in death because of widespread dissemination . The reported overall survival rates without local recurrence are 30% at 5 years and 16% at 10 years . Some patients experience rapidly fatal progression, and late metastasis after many years of freedom from disease is also relatively common . We believe that regular long - term follow - up with computed tomography, magnetic resonance imaging, and 18f - fluorodeoxyglucose positron emission tomography examination is important to identify local recurrence or distant metastasis as early as possible.
A well - established theoretical framework for the evolution of cooperative interactions contrasts partner fidelity and partner choice as the most important mechanisms promoting and maintaining cooperation . Previous studies have empirically shown that partner choice via host sanctions or differential rewards can stabilize cooperation in environmentally transmitted symbioses like mycorrhizal fungi or nitrogen - fixing rhizobia of plants, while the specialized intracellular symbioses of insects are generally assumed to be stabilized by partner fidelity . It remains unknown, however, which factors contribute to the maintenance and specificity of the vast majority of symbiotic associations in animals that involve facultative microbial associates, which contribute significantly to the ecological success of insects as well as many other organisms . Beewolves are solitary digger wasps of the genera philanthus, trachypus, and philanthinus (hymenoptera, crabronidae) that hunt other hymenoptera and provision them as prey for their developing offspring in subterranean brood cells . Female beewolves cultivate symbiotic bacteria in specialized gland reservoirs in the antennae and secrete them into the brood cells prior to oviposition . The larvae later transfer the symbionts to the cocoon silk, where they provide protection during subsequent development which is often not completed until the following year by producing a mixture of at least 9 different antimicrobial substances . Recent phylogenetic analyses indicated that the symbionts are descendants of soil - dwelling streptomycetes that were acquired by the insects at least 68 million years ago (fig . The external route of vertical symbiont transmission from mothers to daughters resulted in host - symbiont co - diversification, but also allowed for horizontal exchange of symbionts among hosts . Artificial infection of beewolf females with opportunistic soil bacteria revealed that these bacteria can grow in the antennal reservoirs but are not transmitted to the offspring, providing strong evidence for partner choice via host control over symbiont transmission . We previously discussed the importance of partner choice for the long - term stability of the beewolf - streptomyces mutualism and will focus here on the implications of its biogeographic history . Node ages in the host phylogeny (left) are shown in million years ago (mya) with 95% highest posterior density (hpd) interval bars . Values at the nodes of the symbiont phylogeny (right) are local support values from the fasttree analysis (gtr model), bootstrap values from phyml, and bayesian posteriors, respectively . Branches are color - coded according to the geographic distribution of the host species (see world map, hatched yellow and red branches indicate occurrence in africa and/or eurasia). Colored boxes around host and symbiont names denote host genera (green = philanthinus, blue = philanthus, red = trachypus). Node ages in the host phylogeny (left) are shown in million years ago (mya) with 95% highest posterior density (hpd) interval bars . Values at the nodes of the symbiont phylogeny (right) are local support values from the fasttree analysis (gtr model), bootstrap values from phyml, and bayesian posteriors, respectively . Branches are color - coded according to the geographic distribution of the host species (see world map, hatched yellow and red branches indicate occurrence in africa and/or eurasia). Colored boxes around host and symbiont names denote host genera (green = philanthinus, blue = philanthus, red = trachypus). The reconstruction of the beewolf phylogeny and a calibration based on the fossil record allows for proposing hypotheses on the biogeographic history of these solitary wasps and their symbiotic association with streptomyces . However, as the fossil record underlying the dating analyses is rather sparse (2 philanthini, one cercerini, and one bembicinae fossil), age estimates are characterized by broad confidence intervals, so the exact timing of biogeographic events remains speculative . Nevertheless, the phylogeny of philanthinae reveals eurasia or africa as the probable origin of beewolves, because philanthinus and the oldest nodes in philanthus all have palearctic or paleotropical distributions (fig . The radiation of beewolves likely followed those of angiosperms and their most important pollinators, the bees, as adult beewolves feed on the nectar of angiosperms and predominantly use bees as larval provisions . Bees originated during the early to middle cretaceous in the southern hemisphere (gondwana), most likely in africa, and the majority of extant philanthus species are african (77 of 137), so an african origin of beewolves seems likely . Interestingly, the south indian philanthus species (philanthus pulcherrimus, philanthus sp . In - e010, and philanthus cf . Basalis) are interspersed among asian and african taxa in the phylogeny, suggesting that the indian subcontinent was colonized from both africa and asia . After spreading across the paleotropics and palearctic, beewolves colonized the americas about 37 mya (95% ci: 2551 mya). As the de geer and thulean bridges broke up around 63 and 56 mya, respectively, colonization via beringia during the warm climate of the late eocene seems most likely, which has previously been suggested for other insect taxa (e.g. Aphids). The colonization of south america may have occurred via the aves ridge or island arc (34 mya), which existed roughly around the time of the estimated split between the south american trachypus and the north american philanthus clade (about 30 mya, 95% ci: 1941 mya, see fig . 1). A plausible scenario assumes a single colonization event with a subsequent radiation of the trachypus clade in south america . However, the current range of trachypus extends north to southern texas, and although it is possible that the northern species have arisen since the emergence of the panamanian land bridge, the phylogenetic relationships of these taxa have yet to be investigated and they may date from an earlier time . Despite a monophyletic origin of the beewolf symbiont clade, it shows many discrepancies with the beewolf phylogeny, indicating frequent horizontal transfer of symbiont lineages among host species . Such transfer could conceivably occur through interspecific predation or nest reuse, or by infection from an environmental reservoir of symbiont spores, all of which require the co - occurrence of a host and its horizontally acquired symbiont . Surprisingly, the symbiont strains show only a moderate degree of clustering according to their hosts' geographical distribution (fig . This pattern is unlikely to be explained by poor resolution or phylogenetic errors, because the phylogeny is based on the sequences of 5 different genes and was recently corroborated by an independent analysis using genome - wide aflp markers . Four mutually non - exclusive hypotheses may explain the occurrence of closely related symbionts in geographically widely separated host taxa: (i) the host taxa colonizing new geographical areas may have carried a mixture of symbionts, which were subsequently exchanged among hosts, and individual symbiont strains were subsequently lost in different host lineages . Although possible, this scenario seems unlikely, as recent analyses suggest a high degree of homogeneity in symbiont populations within individual beewolves . Furthermore, given the ancient separation of old and new world species, biogeographic patterns would still be expected in the symbiont phylogeny under this scenario . (ii) the symbionts may be dispersed via wind or water over large distances (even across continents and oceans) and infect novel hosts . Although recent studies provide increasing evidence for dispersal limitation and biogeographic patterns in microorganisms, some microorganisms indeed appear to be globally dispersed, and dormancy can be expected to facilitate long - range dispersal and successful colonization of new habitats . In fact, trans - oceanic dispersal events of microorganisms in dust clouds have been well documented, particularly from africa to the americas . Previous studies have shown that the beewolf symbionts undergo morphological differentiation on the beewolf cocoon and thereby survive inhospitable conditions as dormant cells for at least 9 months, and possibly much longer . Seed banks of ancient symbiont spores in the environment, thereby obscuring apparent biogeographic patterns among supposedly extant symbiont lineages . (iv) it is conceivable that the symbionts are only facultatively associated with beewolves and occur as free - living bacteria in the environment, which would provide ample opportunities for horizontal transfer and at least partially obscure biogeographic patterns (although isolation by distance would still be expected, if dispersal is limited). Possibly, the symbiotic ancestors were already globally distributed, and subsequent vicariance events and host switches resulted in the diversification of bacterial strains and led to the observed phylogenetic patterns, with closely related symbiont strains occurring in geographically distant host taxa . The recent isolation of closely related streptomyces strains from moroccan soil and from chilli pepper rhizosphere in thailand could provide evidence for the environmental occurrence of the beewolf symbionts, although the phylogenetic affiliation of these isolates with the symbiont clade is currently based on 16s rdna only and needs confirmation by multi - locus sequencing . Beewolves and antibiotic - producing streptomyces bacteria participate in a defensive symbiosis involving both vertical and horizontal symbiont transmission . The biogeographic history of beewolves is well defined, but that of the symbionts is only loosely concordant with it, suggesting global dispersal or vicariance . Could greatly advance our understanding of the biogeographic history of the ancient association between beewolves and streptomyces, and thereby illuminate larger questions about the relative importance of animal - vectored and free - living dispersal in shaping the distribution of microorganisms in nature . We gratefully acknowledge financial support from the max planck society (mk) and the german science foundation (dfg - str532/2 - 2 [es / mk] and dfg - ka2846/2 - 1 [mk]).
Preoperative anxiety or stress have been thought to delay gastric emptying and increase gastric acidity and is therefore considered one of risk factors related with aspiration pneumonitis . In large series of olsson et al . (1), it was reported that pain and anxiety were able to be important mechanisms of decreasing intestinal motility especially in emergency surgical cases . Similarly, outpatients tend to have increased gastric fluid, presumably because of anxiety (2). Cote et al . (3) found that more anxious children had a higher gastric acid content, although the difference was of questionable clinical significance . More recently, clinical studies using various methods, however, have failed to confirm any correlation between anxiety and gastric function (4 - 6). The purpose of this study was to clarify whether anxiety affects the preoperative actual acidity and volume of gastric fluid with several contributing factors such as sex, age, fasting time, hypoglycemic status controlled in female patients undergoing elective gynecologic surgery under general anesthesia . After obtaining an approval from the committee of ethics on human study in our hospital and an informed consent from each patient, we studied asa physical status i - ii 96 female patients aged between 16 and 60 yr who underwent elective gynecological surgery under general anesthesia . Patients with a history of any gastrointestinal disorder, or who were receiving antacids or h2 receptor blockers, or any other medication, which would interfere with gastrointestinal function, were excluded from the study . A power analysis was performed to determine the sufficient sample size required to establish a significant difference in the gastric variables and in the percentage of patients at risk for aspiration pneumonia based on the results of a preliminary study, using an -value of 0.05, and power of 0.8 ., patients were asked to complete the visual analogue scale with regard to the level of anxiety (range 0=no anxiety at all, to 10=extremely anxious) in the preoperative preparing unit . The patient's data including vital signs, oral fasting time, and medical and surgical history were collected from interview and the medical records . Immediately after tracheal intubation using 100 - 120 mg of propofol and 0.5 mg / kg of rocuronium, a 14-f multiorifice nasogastric tube (levin tube, yushin medical, shiheung, korea) was inserted upto 60 cm from the incisor . Proper position of the nasogastric tube was verified by auscultation over the epigastrium by injecting 10 ml of air . Subsequently, the gastric fluid was aspirated gently to a syringe using mild negative pressure . This maneuver was repeated in both slightly left- and rightward tilted positions and in trendelenburg and reverse trendelenburg position to ensure maximum emptying of the stomach . A standard general anesthesia with enflurane in nitrous oxide, oxygen, and rocuronium was provided for the surgery . The volume of gastric fluid and ph were measured using a metered cylinder and a ph meter twice (model-920a, orion research inc . Blood concentration of glucose was measured at the same time by using a blood glucose meter (surestep, lifescan inc ., milpitas, ca, u.s.a . ). Blood samples taken from the arm opposite to the intravenous infusion were collected and centrifuged; the serum was separated and stored at -70. double antibody gastrin method is a radioimmunoassay technique for gastrin designed for the quantitative measurement of gastrin in serum, which utilizes labeled synthetic iodinated human gastrin (shg i-17) (7). Visual analogue scale (vas) of preoperative anxiety was 3.822.1 and the range was from 0 to 8.5 . We classified the subjects into 2 groups, those presenting vas less than 5 (low - anxiety group, l - group), and remains (high - anxiety group, h - group). Statistical analysis was performed by paired t - test with bonferroni correction for demographic data . The mann - whitney u - test and chi - square test were used to analyze the anxiety scores, gastric ph and volume of the gastric fluid, serum gastrin concentrations, and the number of patients at risk for aspiration pneumonitis (gastric ph <2.5 and volume> 25 ml) in order to compare both groups . Spearman's correlation coefficient was used to measure the association between anxiety and gastric residues . The two groups were comparable with regard to age, weight, height, diagnosis, fasting time, and preoperative blood glucose level . Preoperative heart rate, systolic and diastolic blood pressures were similar in the both groups (table 1). The preoperative anxiety score (vas) of l - group was 2.41.4, and that of h - group was 6.00.9 . The mean gastric acidity and volume of the two groups were not statistically different: ph of l - group, 3.01.8 (range: 1.3 - 7.3), ph of h - group, 3.02.0 (range: 1.3 - 7.8), volume of l - group, 15.311.7 ml (range: 0 - 50), and volume of h - group, 11.811.8 ml (range: 0 - 70). There were 9 (15.3%) patients considered to be' at risk: gastric ph <2.5 and volume> 25 ml' in the l - group, and 1 (2.7%) in the h - group (p<0.05, fig . The mean serum gastrin concentration in the l - group was 21.69.8 pg / ml, and that in the h - group was 20.211.0 pg / ml (p = ns). The ph and volume of preoperative gastric contents were not significantly associated with the preoperative anxiety (fig ., there were no epistaxes, vomiting, ventilatory problem, or other serious complications . The main finding of this study was that there are no differences in preoperative gastric ph and volume between the two groups, but there are more patients' at risk' for aspiration pneumonitis in the low anxiety group . Several authors reported that preoperative anxiety did not prolong gastric emptying nor had influence gastric acidity and volumes in adults and children (3 - 5, 8, 9). (6) evaluated the relationship between oral premedication, preoperative anxiety, and gastric contents in patients having elective surgery . Gastric volume and ph did not correlate with the type of premedication or the level of anxiety . (9) observed that a smaller gastric fluid volume in highly anxious pediatric patients compared to children with low anxiety level . In addition, the increased sympathetic tone disturbs gastric secretion through the adrenergic neurons in the splanchnic nerve . In our results, however, we could not detect any differences in serum gastrin concentration which has been known as a major hormone regulating secretion of gastric juice . Interestingly, we also observed that the number of' patients at risk: gastric ph <2.5 and volume> 25 ml' was much greater in less anxious patients . The smaller gastric volume of highly anxious patients was consistent with the report of kawana et al . Therefore, perhaps gastric motility, not gastric secretion, was altered by neural or endocrine mechanisms in the anxious patients . Traditionally, the -adrenergic system has been shown to exert an inhibitory action on gastric emptying in normal circumstances; isoprenaline decreases and propranolol increases stomach emptying (10, 11). In contrast, christensen and stadil (12) reported that corticosteroids and adrenaline, in physiological concentrations, increase the acidity of gastric secretions . We did not measure hormones including epinephrine, norepinephrine, or any other stress hormones . Although our results did not support any association between the level of preoperative anxiety and gastric volume and ph, preoperative anxiety seems to decrease the risk of aspiration pneumonitis . Further studies are necessary to clarify neurogenic and endocrine regulatory mechanisms of anxiety on preoperative gastric function . Since gastric secretion and movements are controlled by a very complicated mechanism and gastric residuals are affected by many factors, a well controlled study design is difficult to create . We tried to minimize other influencing factors for gastric secretion and emptying such as sex, age, fasting time, preoperative blood glucose concentration, and premedication . No attempt was made to take account of the phase of menstrual cycle or circadian variation because the effect of both has been shown not to apply to gastric emptying of liquids (13, 14). This may be due to environmental factors including unpredictable delay of the operation, or protocol violation by the patient such as skipping the previous evening meal due to preoperative anxiety and tension . The technique of gastric aspiration cannot guarantee complete gastric emptying, and volumes recorded reflect only the maximum gastric aspirate obtained at the time, with every effort made to increase yield by manipulation of the tube . Dye absorption technique and fiberoptic gastroscopy are more reliable than blind aspiration (15), but are more complicated, time consuming, and clinically limited methods . We evaluated the patients' anxiety in terms of vas based on the study of kindler et al . They reported that the vas allows effective measurement of preoperative anxiety and detection of patients' anesthetic concerns . We conclude from the current data that there are no significant differences of preoperative gastric ph and volumes between high and low anxious patients . However, the number of patients' at risk' for aspiration pneumonitis was much more in low anxious patients . The results suggest that a low level of preoperative anxiety can be considered a risk factor in connection with aspiration pneumonitis.
Generally, plastic reconstruction involves free skin grafts (3) or flaps . Soft tissue defects that are present in the diabetic foot often need flap reconstruction . It is our opinion that skin grafts are not usually recommended for a majority of the extremely deep and severe wounds encountered forms b, c, d; grades ii, iii (table 1); and especially when they are located at the weight - bearing aspect of the foot and without enough subdermic soft tissue (fat or muscle) between the skin graft and the underlying bone . Dorsal and some plantar soft tissue defects and non - weight - bearing donor site defects are usually the only diabetic foot wounds we elect to cover with a skin graft . The flaps could be random - local (advancement, transpositional, or rotating) (4) or vascularized . Vascularized flaps could be free, pedicled, fasciocutaneous, or muscle derived . Pedicled fasciocutaneous flaps could be axial reverse or orthrodromic or perforator based . Another, traditional flap that can also be used for the above mentioned cases is the cross leg distant flap . The most effective choice for soft tissue flap coverage typically depends on multiple factors including but not limited to the location, size, appearance, and depth of the wound in conjunction with the vascularity of the limb and the presence of underlying pathology . The location of the defect is described according to the surface (dorsal, plantar, medial, and/or lateral) and the functional character of the injured area (weight - bearing area, peri - articular, non - weight bearing, etc . ). A superficial wound can be treated with surgical debridement, non - weight - bearing, and secondary healing or skin grafting (2). For deeper wounds, local random flaps include transpositional, advancement, and rotational flaps that incorporate the skin, subcutaneous tissue, and sometimes the fascia for transfer . Plantar defects, such as sub - cuboid ulcerations from charcot neuroarthropathy and sub - metatarsal head ulcerations are especially well suited to this type of flap coverage so as to cover like with like tissue . Modifications of bilobed and v - y random advancement flaps are typically utilized for the coverage of sub - metatarsal ulcers and plantar defects as long as no underlying osteomyelitis is present (5, 6). Local intrinsic muscle flaps are another option for closure of plantar weight - bearing wounds or to cover osseous defects after surgical management of osteomyelitis . Most frequently used muscle flaps in the foot are the flexor digitorum brevis, abductor hallucis, abductor digiti minimi, and the extensor digitorum brevis muscles (7). In the diabetic foot, remote pedicle island flaps are commonly utilized for the weight - bearing surface and to restore sensation . Pedicle flaps involve the local transposition of skin, subcutaneous tissues, and the associated neurovascular supply to cover a soft tissue defect and may be designed with retrograde or anterograde vascular inflow . Pedicle flaps are indicated to salvage failed local - random flaps, failed muscle flaps, and for larger soft tissue defects particularly over previous pedal amputations or heel defects . Pre - operative planning for these flaps involves meticulous evaluation of the vascular supply and its anatomic variations . The most common pedicle flaps utilized in the diabetic foot include the digital artery flap, medial and lateral plantar artery flaps, and reverse flow sural artery flap . In cases of using a vascularized flap (pedicled or free), it is preferred to utilize a neurovascular pedicle flap (medial plantar artery flap or reverse flow sural artery flap) if feasible and in order to restore sensation on the weight - bearing surface (fig . 1 and fig . The management of diabetic foot and ankle soft tissue defects must be based on the safer flap according to the vascularity of the limb, patient's co - morbidities and if a flap failure occurs, a more complicated flap could follow . For example, if closure with a local - random flap is feasible, it should be attempted first . Free tissue transfers with vascular anastomosis may be performed with skepticism and after a thorough evaluation of the vascular status of the diabetic limb with angiography . Free tissue transfer utilized for complex diabetic foot and/or ankle wounds typically require harvest of the latissimus dorsi or gracillis muscle with microvascular anastomosis to a patent artery of the lower extremity (8, 9) (fig . Regardless of the plastic surgery reconstruction chosen, numerous complications are possible, with flap necrosis being the most common . Flap necrosis is classified as partial / superficial or full thickness (fig . 4 and fig . 5). The causes for flap necrosis are numerous and need to be understood particularly when attempting to salvage a flap that developed necrosis . Additionally, patient - related factors in the diabetic population contribute significantly to flap complications; therefore careful patient selection and co - management of the patient's co - morbidities is of utmost importance . Technical errors such as compromise to the angiosome, pedicle, or vascular anastomosis and excessive tension on the flap should be avoided in order to decrease the chances of flap ischemia and necrosis . Meticulous hemostasis is paramount to prevent hematoma formation that can lead to venous congestion and flap necrosis . In addition, addressing pre - existing conditions such as osteomyelitis and vascular disease often have to be re - evaluated to determine if further intervention are required that may be jeopardizing the overlying flap . Regardless of the circumstances, the patient should be monitored closely in the postoperative setting so that complications can be recognized and treated early . Frequently, local wound care, hyperbaric oxygen therapy, adequate off - loading, as well as continuance of antibiotic therapy might be necessary during the patient's recovery period . Clinical view of superficial necrosis of a reverse flow the goal of revisional surgical treatment for failed soft tissue coverage is to eradicate infection if present, reassess and address any vascular compromise to the extremity, re - evaluate the underlying osseous structure and correct it if needed, and perform delayed soft tissue coverage when all of the previous factors are addressed . Deciding which patients are proper candidates for revisional and reconstructive surgery depends on the above mentioned factors . Before performing a second plastic reconstruction or another type of treatment, the surgeon needs to address the failure reasons that lead to the flap complication . A multidisciplinary team approach is taken to ensure optimization of the patient's systemic condition(s). Glycemic control is extremely important and must be addressed by the internists and/or endocrinologists so that blood sugars are normalized during the peri - operative period . If patient non - compliance is the primary cause for the flap failure and patient education with eventual compliance cannot be established, the patient may be better served with amputation of the affected extremity or continuation of prolonged wound care modalities . The patient's peripheral circulation needs to be thoroughly evaluated when flap necrosis and soft tissue loss is evident . The necessity of immediate vascular work up consisting of non - invasive and invasive vascular studies is performed to determine whether conservative vascular intervention through the administration of vasodilators and/or anticoagulation is sufficient . For more complicated arterial occlusions, endovascular intervention, arterial - venous bypass with a saphenous vein, sympathectomy, and/or lower extremity bypass is required . It is also paramount to understand the importance of emergent diabetic foot surgery in the presence of a severe limb ischemia . However, vascular surgery should be consulted as early as possible especially in the face of flap compromise so that both disciplines can reach a consensus on the final treatment plan and to also perform any needed revascularization and revisional plastic surgery to obtain successful diabetic limb salvage . Delayed reconstructive plastic surgery procedures need to be coordinated with the vascular team to determine the best time for a definitive soft tissue closure of the diabetic foot . Once the patient is optimized, a hierarchy of available options for soft tissue reconstruction in the diabetic foot is applied based on the size and location of the defect while considering the vascularity of the limb and what available local tissue can be utilized . If failure of a skin graft is observed, it is usually because the graft was too thin and/or hematoma, seroma or infection had developed . If skin graft failure was because of placement on an osseous prominence or on a weight - bearing aspect of the foot, then salvage is usually performed with a local random flap if the surrounding tissues are sufficient and a vascularized pedicle or perforator flap if they are not . In the event of failure of a local random flap, a pedicle or perforator flap can be utilized . The pedicle or perforator flaps of the lower extremity can also be utilized to salvage failure of a free vascularized flap and should be attempted, if feasible, before performing a revisional free vascularized flap . Another option that exists is to utilize a pedicle flap from the contralateral extremity, the cross - leg flap, if options are limited . In addition, a combination of flaps can also be utilized including a local random flap combined with a pedicle or perforator flap to cover larger soft tissue defects . The modified papineau technique (10, 11) provides another alternative for management of both soft tissue and bone defects especially in recurrent osteomyelitis in diabetic severe lesions (forms b, c, d and grades ii, iii) (1). This technique allows for secondary wound healing and has been described with increased popularity over the last decade, as a solution for persistent osteomyelitis and as a salvage procedure . The modified papineau open grafting procedure (10) consists of a radical treatment procedure of the recurrent osteomyelitis following severe lower limb trauma or infection and the secondary reconstruction of bone and soft tissue defects without closing the skin when other procedures have already failed (bone grafting, negative pressure wound therapy, and a free or regional flap performance) after, of course, a meticulous debridement and curettage . While in the classical papineau technique the surgeon uses corticocancellous bone chips, our modifications consist of the use of only cancellous bone that is harvested from the iliac crest with a minimal invasion technique . The modified papineau procedure is not the first treatment choice but a salvage solution, which in some cases, is very effective . As with any other surgical technique, it is imperative to perform a meticulous debridement and resection of the septic osseous segment before performing the papineau technique . This papineau technique is utilized often when major free tissue transfer or pedicle flaps are not feasible as a final attempt for salvage prior to amputation . The frequent co - morbidities in this patient population necessitate careful pre - operative planning and a multidisciplinary approach for optimal outcomes . A hierarchy of available options for soft tissue reconstruction in the diabetic foot is applied based on the size and location of the defect in conjunction with the vascularity of the limb . The authors have not received any funding or benefits from industry to conduct this study.
Severe chronic neutropenia is a congenital condition defined as an absolute neutrophil count (anc) of less than 500/l for at least three months (1, 2). Generally, underlying hematologic disorders, autoimmune diseases, viral infections, or drugs causing neutropenia should be excluded . There are three categories according to the onset time or pattern of variation of neutrophil levels: congenital, cyclic, and idiopathic . Among them, severe congenital neutropenia (scn) is an inborn disorder with maturation arrest of the early stage of granulopoiesis associated with various genetic abnormalities (3). In addition, cyclic neutropenia (cn) is a related disorder characterized by periodic oscillation of the peripheral neutrophil count, with an average 21-day frequency (1, 2). Among several associated genetic mutations, the variants of the elane sequence comprise approximately 50% of the genetic causes of scn and nearly all of cn (4), and it is known to be correlated with more severe neutropenia and serious clinical manifestations in scn (5). We herein introduce a korean girl with typical features of scn and a novel elane gene mutation . A 9-month - old girl who was born on september 17th, 2009 was transferred to our hospital with prolonged fever and recurrent cervical lymphadenitis on june 25th, 2010 . Her initial laboratory tests revealed severe neutropenia (anc 90/l) and increased acute phase reactants (erythrocyte sedimentation rate 97 mm / hr and c - reactive protein 7.9 mg / dl). She had a past history of admission to the neonatal intensive care unit at another hospital at birth due to omphalitis . Her course of development was normal, and she had no congenital malformation suggestive of specific syndromes associated with neutropenia . The cervical contrast enhanced computed tomography (ct) was performed to exclude abscess formation or deep neck infection that would need incision or aspiration . Bone marrow (bm) findings showed early - stage maturation arrest of myelopoiesis (cellularity 80%-100%, myeloblasts 3.0%, promyelocytes 0.7%, myelocytes 2.2%, metamyelocytes 0.5%, band neutrophils 0.2%, and segmented neutrophils 0.0%), with an m: e ratio of 0.43:1 (fig . Considering her past history and physical examination, scn associated with elane abnormality was suspected . Direct dna sequencing analysis of the elane gene demonstrated a substitution of the 607th base (g to c) in exon 5, resulting in a change of the 203rd codon (glycine to arginine), which is a novel variation of scn (c.607 g> c; p.gly203arg) (fig . She had no increase in her anc despite daily administration of subcutaneous 5 - 10 g / kg granulocyte colony stimulating factor (g - csf). After management with an increased dose (15 g / kg / day) of g - csf for six consecutive days, she recovered and was discharged with a increased anc (11,110/l) (fig . However, the anc was again decreased at 990/l when she visited our outpatient clinic one week after cessation of the g - csf injections, and reduced to 130/l after one month . She has suffered subsequent seven episodes of febrile illnesses until the present (table 1). All of them were managed with prompt administration of intravenous empiric broad - spectrum antibiotics, cefepime . Scn is a very rare condition that is diagnosed when the anc is less than 500/l from birth, with myeloid cell differentiation arrest at an early stage in the bm (1, 3). Cn is also a congenital disorder of granulopoiesis characterized by periodic oscillations in the number of circulating neutrophils (1, 2). Patients experience infections when the neutrophil count drops below 500/l and approaches zero . Both scn and cn are related to an elane alteration associated with sporadic or autosomal dominant inheritance (6). Scn is a genetically heterozygous disease group with several gene mutations and various modes of inheritance (3, 7). For example, patients with the hax1 abnormality show developmental delay or epilepsy with autosomal recessive inheritance (kostmann syndrome), while patients with the elane mutation, which arises sporadically or follows autosomal dominant inheritance, have no neurological problems . Her diagnosis was delayed, however, until nine months of age, because her past history of an umbilical cord infection and isolated severe neutropenia was overlooked . It is important to reconsider patients' history and laboratory tests in the case of recurrent bacterial infections . Since the patient had no anomalous morphology or neurological problems, we first suspected the elane gene mutation . If the patients show any malformations, fanconi anemia / schwachman - diamond syndrome (skeletal abnormality), dyskeratosis congenita (nail dystrophy), or diamond - blackfan syndrome (anomaly of face or hand) should be considered (8). Mutation of the elane gene (chromosome 19p13.3) encoding human neutrophil elastase (ne) is the most common genetic alteration in scn (3). It comprises about 50% of scn and nearly all of cn (3, 4). There are some cases of korean scn patients, but until today, a genetically confirmed elane gene mutation was the only one in korea (c.170c> t; p.ala57val) (9). Neutrophils play a key role in innate immunity and the host's defense mechanisms by phagocytosing, killing, and digesting bacteria and fungi (10). In particular, azurophilc (primary) granules in the neutrophil, which are formed during the promyelocytic stage, contain several important proteins (myeloperoxidase, cathepsin g, elastase, proteinase 3, bactericidal / permeability - increasing protein, and defensin) essential for antibacterial activity . Among them, ne attacks gram negative bacteria like klebsiella pneumoniae or escherichia coli (11). In neutrophils in which ne is inactivated, bacteria escape from the phagosome, resulting in increase of bacterial survival (12). Although the entire pathophysiology of how the elane mutation develops in neutropenia is unknown, recently it is believed that the mutant ne tends to be misfolded, which leads to apoptosis of myeloid progenitor cells through an' unfolded protein response' by the endoplasmic reticulum (13). For those reasons, there are few mature neutrophils, while promyelocytes are normally present; that is the so - called' maturation arrest' in the bm, and thus patients suffer from lifelong infections in scn . Morphologically prominent vacuolations or aberrations of azurophilc granules may appear in the bm (7). Infections include bacterial illnesses, such as a urinary tract infection, pneumonia, omphalitis, septicemia, lymphadenitis, otitis media, cellulitis, and fungal disease like oral thrush . Neutrophils contribute to tissue injury and reconstitution as well as play a role in host - defense cells (14). Thus, absence of pus at infection sites is the characteristic feature of scn, as demonstrated by the neck ct of the patient in this study (7). As a treatment of choice, the severe chronic neutropenia international registry recommends that the g - csf injection is started at a dose of 5 g / kg / day and continued in upward 2-fold adjustments until the anc reaches over 1,000 - 1,500/l (15). Generally, scn patients require a higher dose of g - csf (median 7.3 - 23.0 g / kg / day, maximum 242.4 g / kg / day) compared to cyclic or idiopathic neutropenia patients . About 90% of scn patients respond to g - csf treatment, but the remainder have only a temporary or no response to g - csf (16). Although the predictors of poor response are not yet demonstrated, most refractory cases have very low initial anc levels . In addition, the elane gene mutation is known to be associated with more severe neutropenia and serious clinical manifestations in scn (5). Although septic mortality is markedly decreased by g - csf treatment, the cumulative incidence of myelodysplastic syndrome (mds) or acute myeloid leukemia (aml) in scn is about 20% after 10 yr treatment (17). It is controversial whether scn is an obvious precancerous condition, or the g - csf treatment contributes to evolution to mds / aml . Considering that cyclic or idiopathic neutropenia patients do not develop mds / aml in spite of g - csf administration, and refractory scn patients who were exposed to a higher dose of g - csf tend to develop mds / aml, a combination of both theories is also possible . In the case of non - responders, stem cell transplantation from an hla - identical sibling is the optimal therapeutic modality (8, 18). In this case, the patient showed only temporary response to subcutaneous 15 g / kg / day of g - csf for six consecutive days . We have not yet increased the dose of g - csf due to a financial problem associated with standards of korean medical insurance . To control infections, we selected the 4th generation cephalosporine (cefepime) empirically to cover both gram positive and negative bacteria including pseudomonas aeruginosa . Since she has no siblings and is very young, we closely follow her, with the idea of stem cell transplantation in reserve.

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